Publications by authors named "Sasidharan Padmaja Divya"

25 Publications

  • Page 1 of 1

PD-L1 Is an Independent Prognostic Marker in Middle Eastern PTC and Its Expression Is Upregulated by Mutation.

Cancers (Basel) 2021 Feb 1;13(3). Epub 2021 Feb 1.

Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia.

PD-L1 inhibition is a promising therapeutic target whose efficacy has been demonstrated in several cancers. Immunohistochemistry was performed to assess PD-L1 protein expression in PTC. We further conducted in vitro analysis to investigate the role of PD-L1 in regulating in PTC cell lines. PD-L1 over-expression was noted in 32.4% (473/1458) of cases and significantly associated with aggressive clinico-pathological parameters. Importantly, PD-L1 was found to be an independent poorer prognostic marker. We also found PD-L1 to be significantly associated with mutation and patients with co-existing PD-L1 over-expression and mutation had a poor disease-free survival compared to patients with mutation alone. In vitro analysis showed high expression of PD-L1 in -mutated PTC cell lines compared to a wild-type cell line. Inhibition of BRAF using vemurafenib induced PD-L1 expression in -mutated cell lines without affecting cell growth. Knockdown of PD-L1 in -mutated cell lines significantly decreased the cell growth and induced apoptosis. Our data suggest that PD-L1 might represent a useful prognostic marker in Middle Eastern PTC and PD-L1 inhibition could be a potential therapeutic option for aggressive PTC cancers, such as the tall cell variant, mutation-positive patients that are unresponsive to standard treatment.
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http://dx.doi.org/10.3390/cancers13030555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867170PMC
February 2021

Prognostic Value and Function of KLF5 in Papillary Thyroid Cancer.

Cancers (Basel) 2021 Jan 7;13(2). Epub 2021 Jan 7.

Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia.

The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.
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http://dx.doi.org/10.3390/cancers13020185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825749PMC
January 2021

Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer.

Front Pharmacol 2020 3;11:598880. Epub 2020 Dec 3.

Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. Despite current therapeutic and surgical options, advanced EOC shows poor prognosis. Identifying novel molecular therapeutic targets is highly needed in the management of EOC. Krupple-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in a variety of cancer types. However, its role and expression in EOC is not fully illustrated. Immunohistochemical analysis was performed to assess KLF5 protein expression in 425 primary EOC samples using tissue microarray. We also addressed the function of KLF5 in EOC and its interaction with signal transducer and activator of transcription 3 (STAT3) signaling pathway. We found that KLF5 overexpressed in 53% (229/425) of EOC samples, and is associated with aggressive markers. Forced expression of KLF5 enhanced cell growth in low expressing EOC cell line, MDAH2774. Conversely, knockdown of KLF5 reduced cell growth, migration, invasion and progression of epithelial to mesenchymal transition in KLF5 expressing cell lines, OVISE and OVSAHO. Importantly, silencing of KLF5 decreased the self-renewal ability of spheroids generated from OVISE and OVSAHO cell lines. In addition, downregulation of KLF5 potentiated the effect of cisplatin to induce apoptosis in these cell lines. These data reveals the pro-tumorigenic role of KLF5 in EOC and uncover its role in activation of STAT3 signaling pathway, suggesting the importance of KLF5 as a potential therapeutic target for EOC therapy.
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http://dx.doi.org/10.3389/fphar.2020.598880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793801PMC
December 2020

CHD4 Predicts Aggressiveness in PTC Patients and Promotes Cancer Stemness and EMT in PTC Cells.

Int J Mol Sci 2021 Jan 6;22(2). Epub 2021 Jan 6.

Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211, Saudi Arabia.

Chromodomain-helicase-DNA-binding protein 4 (CHD4), a core subunit of the nucleosome remodeling and deacetylation (NuRD) complex is highly expressed in several cancers. However, its role in the pathogenesis and progression of papillary thyroid carcinoma (PTC) has not been investigated. We investigated the prognostic significance of CHD4 in a large cohort of Middle Eastern PTC patients and explored the functional role of CHD4 in regulating cancer stemness and EMT in PTC cells. CHD4 overexpression was observed in 45.3% (650/1436) of PTCs, and was associated with aggressive clinico-pathological parameters and worse outcome. Functional analysis using PTC cell lines showed that forced expression of CHD4 promoted cell proliferation, spheroid growth, migration, invasion and progression of epithelial to mesenchymal transition (EMT) in PTC cells whereas its knockdown reversed the effect. Methylation of E-cadherin was associated with loss of expression in CHD4 expressing cells, while CHD4 depletion reactivated E-cadherin expression. Most importantly, knockdown of mesenchymal transcriptional factors, Snail1 or Zeb1, attenuated the spheroid growth in CHD4 expressing PTC cells, showing a potential link between EMT activation and stemness maintenance in PTC. These findings suggest that CHD4 might be a promising therapeutic target in the treatment of patients with an aggressive subtype of PTC.
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http://dx.doi.org/10.3390/ijms22020504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825451PMC
January 2021

Retraction Note: Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism.

Sci Rep 2019 11 15;9(1):17001. Epub 2019 Nov 15.

Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY, 40536, USA.

This article has been retracted.
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http://dx.doi.org/10.1038/s41598-019-53858-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858308PMC
November 2019

APC truncating mutations in Middle Eastern Population: Tankyrase inhibitor is an effective strategy to sensitize APC mutant CRC To 5-FU chemotherapy.

Biomed Pharmacother 2020 Jan 5;121:109572. Epub 2019 Nov 5.

Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia. Electronic address:

Colorectal Cancer (CRC) is highly heterogeneous for which prognosis is dependent mainly on clinical staging. There is a need to stratify subpopulations of CRC on molecular basis to better predict outcome and therapy response. Truncating mutations in adenomatous polyposis coli (APC) are well-described events in CRC carcinogenesis. Clinical and genotypic characterization of Middle Eastern CRC based on presence and type of APC was determined in 412 CRC tumors using modern next generation sequencing. APC truncating mutations were identified in 58.2% (240/412) of CRCs. Overall, mutation was significant predictor of superior overall survival. Further, the type of APC mutations (short or long) did not have impact on clinical outcome. However, in vitro analysis showed difference between CRC cell lines carrying short truncating APC vs CRC cells that carry long truncating APC mutation in response to 5-flourouracil (5-FU). Importantly, we were able to overcome the resistance to 5-FU seen in CRC cells carrying short APC by tankyrase inhibitor, XAV939, thereby inhibiting Wnt/β-catenin signaling cascade. Overall, our results showed that APC mutation status plays an important role in predicting overall survival in Middle Eastern population. Furthermore, in vitro data showed that selective targeting of APC mutated CRC by tankyrase inhibitor can be an effective strategy to overcome 5-FU resistance in CRC cells.
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http://dx.doi.org/10.1016/j.biopha.2019.109572DOI Listing
January 2020

TGFβ-induced SMAD4-dependent Apoptosis Proceeded by EMT in CRC.

Mol Cancer Ther 2019 07 3;18(7):1312-1322. Epub 2019 May 3.

Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

Colorectal cancer is one of the leading causes of cancer-related deaths worldwide. In Saudi Arabia, colorectal cancer is more aggressive and presents at younger age, warranting new treatment strategies. Role of TGFβ/Smad4 signaling pathway in initiation and progression of colorectal cancer is well documented. This study examined the role of TGFβ/Smad4 signaling pathway in a large cohort of Saudi patients with colorectal cancer, followed by analysis to dissect the dual role of TGFβ on inducing epithelial-to-mesenchymal transition (EMT) and apoptosis. Our study demonstrated high frequency of alterations with low expression of Smad4 protein identifying a subgroup of aggressive colorectal cancer to be an independent marker for poor prognosis. Functional studies using colorectal cancer cells show that TGFβ induces Smad4-dependent EMT followed by apoptosis. Induction of mesenchymal transcriptional factors, Snail1 and Zeb1, was essential for TGFβ-induced apoptosis. Our results indicate that KLF5 acts as an oncogene in colorectal cancer cells regardless of Smad4 expression and inhibition of KLF5 is requisite for TGFβ-induced apoptosis. Furthermore, TGFβ/Smad4 signal inhibits the transcription of KLF5 that in turn switches Sox4 from tumor promoter to suppressor. A high incidence of alterations were found in the Saudi patients with colorectal cancer. Functional study results indicate that TGFβ induces Smad4-dependent EMT followed by apoptosis in colorectal cancer cells.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1378DOI Listing
July 2019

Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment.

Oncotarget 2018 Dec 18;9(99):37319-37332. Epub 2018 Dec 18.

Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

Patients with aggressive breast cancer (BC) subtypes usually don't have favorable prognosis despite the improvement in treatment modalities. These cancers still remain a major cause of morbidity and mortality in females. This has fostered a major effort to discover actionable molecular targets to treat these patients. Poly ADP ribose polymerase (PARP) is one of these molecular targets that are under comprehensive investigation for treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not yet been explored. Therefore, we examined the expression of PARP protein in a large cohort of over 1000 Middle Eastern BC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. Nuclear PARP overexpression was observed in 44.7% of all BC cases and was significantly associated with aggressive clinico-pathological markers. Interestingly, nuclear PARP overexpression was an independent predictor of poor prognosis. PARP overexpression was also directly associated with XIAP overexpression, with PARP and XIAP co-expression in 15.8% (159/1008) of our cases. We showed that combined inhibition of PARP by olaparib and XIAP by embelin significantly and synergistically inhibited cell growth and induced apoptosis in BC cell lines. Finally, co-treatment of olaparib and embelin regressed BC xenograft tumor growth in nude mice. Our results revealed the role of PARP in Middle Eastern BC pathogenesis and prognosis. Furthermore, our data support the potential clinical development of combined inhibition of PARP and XIAP, which eventually could extend the utility of olaparib beyond BRCA deficient cancer.
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http://dx.doi.org/10.18632/oncotarget.26470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324669PMC
December 2018

FoxM1 and β-catenin predicts aggressiveness in Middle Eastern ovarian cancer and their co-targeting impairs the growth of ovarian cancer cells.

Oncotarget 2018 Jan 16;9(3):3590-3604. Epub 2017 Dec 16.

Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.

Epithelial ovarian cancer (EOC) is a highly lethal disease with poor prognosis especially in advanced stage tumor. Emerging evidence has reported that aberrant upregulation of FoxM1 and β-catenin are closely associated with aggressiveness of human cancer. However, interplay between these factors in the aggressiveness of EOC is not fully illustrated. In this study, we show that FoxM1 is frequently increased in Middle Eastern EOC and associated with high proliferative index ( = 0.0007) and high grade tumor ( = 0.0024). Interestingly, FoxM1 is significantly associated with elevated nuclear β-catenin and the concomitant increase of FoxM1 and β-catenin is associated with advanced stage of EOC by immunohistochemical analysis of 261 samples of Saudi patients with EOC. Functional analysis showed that β-catenin is a direct transcriptional target of FoxM1 in EOC cell lines. FoxM1 inhibition either by specific inhibitor, thiostrepton or siRNA suppressed β-catenin expression, whereas overexpression of FoxM1 increased nuclear β-catenin expression. We identified two FoxM1 binding sites in the β-catenin promoter that specifically bound to FoxM1 protein. Down-regulation of FoxM1 using thiostrepton induced apoptosis and inhibited cell migration/invasion in EOC cells. Moreover, co-inhibition of FoxM1 by thiostrepton and β-catenin by FH535 significantly and synergistically inhibited EOC cell growth and . Collectively, our findings confer that co-targeting FoxM1/β-catenin signaling cascade may be a promising molecular therapeutic choice in advanced EOC.
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http://dx.doi.org/10.18632/oncotarget.23338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790485PMC
January 2018

Downregulation of SKP2 in Papillary Thyroid Cancer Acts Synergistically With TRAIL on Inducing Apoptosis via ROS.

J Clin Endocrinol Metab 2018 04;103(4):1530-1544

Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.

Context And Objective: S-phase kinase protein 2 (SKP2) is an F-box protein with proteasomal properties and has been found to be overexpressed in a variety of cancers. However, its role in papillary thyroid cancer (PTC) has not been fully elucidated.

Experimental Design: SKP2 expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of >1000 PTC samples. In vitro and in vivo studies were performed using proteasome inhibitor bortezomib and proapoptopic death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) either alone or in combination on PTC cell lines.

Results: SKP2 was overexpressed in 45.5% of PTC cases and was significantly associated with extrathyroidal extension (P = 0.0451), distant metastasis (P = 0.0435), and tall cell variant (P = 0.0271). SKP2 overexpression was also directly associated with X-linked inhibitor of apoptosis protein overexpression (P < 0.0001) and Bcl-xL overexpression (P = 0.0005) and inversely associated with death receptor 5 (P < 0.0001). The cotreatment of bortezomib and TRAIL synergistically induced apoptosis via mitochondrial apoptotic pathway in PTC cell lines. Furthermore, bortezomib and TRAIL synergistically induced reactive oxygen species (ROS) generation and caused death receptor 5 upregulation through activation of the extracellular signal-regulated kinase-C/EBP homologous protein signaling cascade. Finally, bortezomib treatment augmented the TRAIL-mediated anticancer effect on PTC xenograft tumor growth in nude mice.

Conclusion: These data suggest that SKP2 is a potential therapeutic target in PTC and that a combination of bortezomib and TRAIL might be a viable therapeutic option for the treatment of patients with aggressive PTC.
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http://dx.doi.org/10.1210/jc.2017-02178DOI Listing
April 2018

Telomerase reverse transcriptase mutations are independent predictor of disease-free survival in Middle Eastern papillary thyroid cancer.

Int J Cancer 2018 05 29;142(10):2028-2039. Epub 2017 Dec 29.

Human Cancer Genomic Research, King Faisal Specialist Hospital and Research Centre, , Riyadh, 11211, Saudi Arabia.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. Tumor recurrence occurs in ∼20% of PTCs and some reach advanced stages. Promoter mutation in the telomerase reverse transcriptase (TERT) gene is identified to be a prognostic marker in PTC. However, the contribution of TERT promoter mutation to cancer progression in PTC patients is still not fully understood. In this study, we investigated the incidence of TERT promoter mutations and TERT protein expression and their association with clinicopathological outcomes in a large cohort of PTC samples using direct sequencing technology and immunohistochemistry. Furthermore, two PTC cell lines were utilized to investigate role of TERT mutations in mediating metastasis. Two promoter hotspot mutations C228T and C250T were identified in 18.0% (167/927) of our cohort and were significantly associated with poor 5 years disease-free survival and distant metastasis of PTC. TERT protein overexpression was noted in 20.1% of our PTC cohort and was significantly associated with poor prognostic markers such as older age, extrathyroidal extension and Stage IV tumors. A significant association was also found between TERT overexpression and epithelial-mesenchymal transition (EMT) markers. Functional analysis showed that TERT inhibition reduced cell growth, invasion, migration and angiogenesis in PTC via suppression of EMT in PTC cells. Our results suggest that TERT promoter mutation is an independent predictor of disease-free survival and might drive the metastasis, and downregulation of TERT could potentiate antitumor and antimetastatic activities in PTC.
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http://dx.doi.org/10.1002/ijc.31225DOI Listing
May 2018

Nuclear factor erythroid 2-related factor 2 enhances carcinogenesis by suppressing apoptosis and promoting autophagy in nickel-transformed cells.

J Biol Chem 2017 05 22;292(20):8315-8330. Epub 2017 Mar 22.

Center for Research on Environmental Disease; Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536-0305. Electronic address:

Nickel-containing compounds are widely used in industry. Nickel is a known human carcinogen that primarily affects the lungs. Proposed mechanisms of nickel-induced carcinogenesis include disruption of cellular iron homeostasis, generation of reactive oxygen species (ROS), and induction of hypoxia signaling. However, the precise molecular mechanisms of nickel-induced malignant transformation and tumor development remain unclear. This study shows that the transcription factor Nrf2 is highly expressed in lung tumor tissue and in nickel-transformed human lung bronchial epithelial BEAS-2B cells (NiT cells). Additionally, constitutively high levels of Nrf2 play a critical role in apoptosis resistance in NiT cells. Basal ROS levels were extremely low in NiT cells and were correlated with elevated expression levels of both antioxidant enzymes ( catalase and superoxide dismutases) and antiapoptotic proteins ( Bcl-2 and Bcl-xL). These processes are tightly controlled by Nrf2. Autophagy inhibition, induced pharmacologically or genetically, enhanced Ni-induced apoptosis, indicating that the induction of autophagy is the cause of apoptosis resistance in NiT cells. Using similar approaches, we show that in NiT cells the inhibition of apoptosis decreases autophagy. We have shown that Stat3, which is up-regulated by Nrf2, controls autophagy induction in NiT cells. Colony formation and tumor growth were significantly attenuated by knockdown of Nrf2 or Bcl-2. Taken together, this study demonstrates that in NiT cells constitutively high Nrf2 expression inhibits apoptosis by up-regulating antioxidant enzymes and antiapoptotic proteins to increase autophagy via Stat3 signaling. These findings indicate that the Nrf2-mediated suppression of apoptosis and promotion of autophagy contribute to nickel-induced transformation and tumorigenesis.
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http://dx.doi.org/10.1074/jbc.M116.773986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437238PMC
May 2017

Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism.

Sci Rep 2016 11 23;6:37227. Epub 2016 Nov 23.

Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA.

Arsenic is a well-documented human carcinogen. The present study explored the role of the onco-miR, miR-21 and its target protein, programmed cell death 4 (PDCD4) in arsenic induced malignant cell transformation and tumorigenesis. Our results showed that treatment of human bronchial epithelial (BEAS-2B) cells with arsenic induces ROS through p47, one of the NOX subunits that is the key source of arsenic-induced ROS. Arsenic exposure induced an upregulation of miR-21 expression associated with inhibition of PDCD4, and caused malignant cell transformation and tumorigenesis of BEAS-2B cells. Indispensably, STAT3 transcriptional activation by IL-6 is crucial for the arsenic induced miR-21 increase. Upregulated miR-21 levels and suppressed PDCD4 expression was also observed in xenograft tumors generated with chronic arsenic exposed BEAS-2B cells. Stable shut down of miR-21, p47 or STAT3 and overexpression of PDCD4 or catalase in BEAS-2B cells markedly inhibited the arsenic induced malignant transformation and tumorigenesis. Similarly, silencing of miR-21 or STAT3 and forced expression of PDCD4 in arsenic transformed cells (AsT) also inhibited cell proliferation and tumorigenesis. Furthermore, arsenic suppressed the downstream protein E-cadherin expression and induced β-catenin/TCF-dependent transcription of uPAR and c-Myc. These results indicate that the ROS-STAT3-miR-21-PDCD4 signaling axis plays an important role in arsenic -induced carcinogenesis.
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http://dx.doi.org/10.1038/srep37227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120334PMC
November 2016

Different roles of ROS and Nrf2 in Cr(VI)-induced inflammatory responses in normal and Cr(VI)-transformed cells.

Toxicol Appl Pharmacol 2016 09 26;307:81-90. Epub 2016 Jul 26.

Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA. Electronic address:

Hexavalent chromium (Cr(VI)) is classified as a human carcinogen. Cr(VI) has been associated with adenocarcinomas and squamous cell carcinoma of the lung. The present study shows that acute Cr(VI) treatment in human bronchial epithelial cells (BEAS-2B) increased inflammatory responses (TNF-α, COX-2, and NF-кB/p65) and expression of Nrf2. Cr(VI)-induced generation of reactive oxygen species (ROS) are responsible for increased inflammation. Despite the fact that Nrf2 is a master regulator of response to oxidative stress, silencing of Nrf2 in the acute Cr(VI) treatment had no effect on Cr(VI)-induced inflammation. In contrast, in Cr(VI)-transformed (CrT) cells, Nrf2 is constitutively activated. Knock-down of this protein resulted in decreased inflammation, while silencing of SOD2 and CAT had no effect in the expression of these inflammatory proteins. Results obtained from the knock-down of Nrf2 in CrT cells are very different from the results obtained in the acute Cr(VI) treatment. In BEAS-2B cells, knock-down of Nrf2 had no effect in the inflammation levels, while in CrT cells a decrease in the expression of inflammation markers was observed. These results indicate that before transformation, ROS plays a critical role while Nrf2 not in Cr(VI)-induced inflammation, whereas after transformation (CrT cells), Nrf2 is constitutively activated and this protein maintains inflammation while ROS not. Constitutively high levels of Nrf2 in CrT binds to the promoter regions of COX-2 and TNF-α, leading to increased inflammation. Collectively, our results demonstrate that before cell transformation ROS are important in Cr(VI)-induced inflammation and after transformation a constitutively high level of Nrf2 is important.
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http://dx.doi.org/10.1016/j.taap.2016.07.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527995PMC
September 2016

Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling.

Oncotarget 2016 Aug;7(32):51193-51210

Center for Research on Environmental Disease, University of Kentucky, Lexington, KY, USA.

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with an increased risk of lung cancer. However, the mechanisms underlying Cr(VI)-induced carcinogenesis remain unclear. MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. Studies have shown that miR-21 exerts its oncogenic activity by targeting the tumor suppressor gene programmed cell death 4 (PDCD4). The present study examined the role of miR-21-PDCD4 signaling in Cr(VI)-induced cell transformation and tumorigenesis. Results showed that Cr(VI) induces ROS generation in human bronchial epithelial (BEAS-2B) cells. Chronic exposure to Cr(VI) is able to cause malignant transformation in BEAS-2B cells. Cr(VI) caused a significant increase of miR-21 expression associated with an inhibition of PDCD4 expression. Notably, STAT3 transcriptional activation by IL-6 is crucial for the Cr(VI)-induced miR-21 elevation. Stable knockdown of miR-21 or overexpression of PDCD4 in BEAS-2B cells significantly reduced the Cr(VI)-induced cell transformation. Furthermore, the Cr(VI) induced inhibition of PDCD4 suppressed downstream E-cadherin protein expression, but promoted β-catenin/TCF-dependent transcription of uPAR and c-Myc. We also found an increased miR-21 level and decreased PDCD4 expression in xenograft tumors generated with chronic Cr(VI)-exposed BEAS-2B cells. In addition, stable knockdown of miR-21 and overexpression of PDCD4 reduced the tumorogenicity of chronic Cr(VI)-exposed BEAS-2B cells in nude mice. Taken together, these results demonstrate that the miR-21-PDCD4 signaling axis plays an important role in Cr(VI)-induced carcinogenesis.
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http://dx.doi.org/10.18632/oncotarget.9967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5239469PMC
August 2016

Quercetin inhibits Cr(VI)-induced malignant cell transformation by targeting miR-21-PDCD4 signaling pathway.

Oncotarget 2017 Aug 17;8(32):52118-52131. Epub 2016 Jun 17.

Center for Research on Environmental Disease, University of Kentucky, Lexington, KY, USA.

Hexavalent chromium [Cr(VI)] is an important human carcinogen associated with pulmonary diseases and lung cancer. Inhibition of Cr(VI)-induced carcinogenesis by a dietary antioxidant is a novel approach. Quercetin is one of the most abundant dietary flavonoids widely present in many fruits and vegetables, possesses potent antioxidant and anticancer properties. MicroRNA-21 (miR-21) is a key oncomiR significantly elevated in the majority of human cancers that exerts its oncogenic activity by targeting the tumor suppressor gene programmed cell death 4 (PDCD4). The present study examined the effect of quercetin on the inhibition of Cr(VI)-induced malignant cell transformation and the role of miR-21-PDCD4 signaling involved. Our results showed that quercetin decreased ROS generation induced by Cr(VI) exposure in BEAS-2B cells. Chronic Cr(VI) exposure induced malignant cell transformation, increased miR-21 expression and caused inhibition of PDCD4, which were significantly inhibited by the treatment of quercetin in a dose dependent manner. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of quercetin showed reduced tumor incidence compared to Cr(VI) alone treated group. Stable knockdown of miR-21 and overexpression of PDCD4 or catalase in BEAS-2B cells suppressed Cr(VI)-induced malignant transformation and tumorigenesis. Taken together, these results demonstrate that quercetin is able to protect BEAS-2B cells from Cr(VI)-induced carcinogenesis by targeting miR-21-PDCD4 signaling.
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http://dx.doi.org/10.18632/oncotarget.10130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5581017PMC
August 2017

Antioncogenic and Oncogenic Properties of Nrf2 in Arsenic-induced Carcinogenesis.

J Biol Chem 2015 Nov 18;290(45):27090-27100. Epub 2015 Sep 18.

Center for Research on Environmental Disease, College of Medicine, University of Kentucky, Lexington, Kentucky 40536-0305; Departments of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, Kentucky 40536-0305. Electronic address:

Arsenic (As(3+)) is a carcinogen with considerable environmental and occupational relevancy. The present study shows that As(3+)-transformed human lung bronchial epithelial BEAS-2B cells (AsT cells) exhibit the property of apoptosis resistance. The level of basal reactive oxygen species (ROS) is very low in AsT cells in correlation with elevated expressions of both antioxidant enzymes and antiapoptotic proteins. Nuclear factor erythroid 2-related factor (Nrf2) and p62 are constitutively expressed. These two proteins up-regulate antioxidant enzymes and antiapoptotic proteins. The knockdown of Nrf2 or p62 by small interfering RNA (siRNA) enhanced both ROS levels and As(3+)-induced apoptosis in transformed cells. AsT cells have autophagy deficiency as evidenced by reduced formation of microtubule-associated protein 1 light chain 3 (LC3)-II, GFP-LC3 puncta, and autophagy flux. Results obtained using a soft agar assay and shRNA Nrf2-transfected cells show that Nrf2 plays an antioncogenic role before transformation, whereas this transcription factor plays an oncogenic role after transformation. In addition, depletion of Nrf2 by shRNA dramatically inhibited growth and proliferation of transformed cells. Furthermore, the Nrf2 protein levels and antiapoptotic and antioxidant enzyme levels are higher in lung adenocarcinoma than in normal tissues. Collectively, this study demonstrates that a constitutively high level of Nrf2 in AsT cells up-regulates the antioxidant proteins catalase and superoxide dismutase as well as the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decreased ROS generation and increased apoptotic resistance, cell survival and proliferation, and tumorigenesis.
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http://dx.doi.org/10.1074/jbc.M115.675371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646382PMC
November 2015

Arsenic Induces Insulin Resistance in Mouse Adipocytes and Myotubes Via Oxidative Stress-Regulated Mitochondrial Sirt3-FOXO3a Signaling Pathway.

Toxicol Sci 2015 Aug 15;146(2):290-300. Epub 2015 May 15.

Department of Toxicology and Cancer Biology, University of Kentucky, 1095 Veterans Drive, Lexington, KY 40536, USA,

Chronic exposure to arsenic via drinking water is associated with an increased risk for development of type 2 diabetes mellitus (T2DM). This study investigates the role of mitochondrial oxidative stress protein Sirtuin 3 (Sirt3) and its targeting proteins in chronic arsenic-induced T2DM in mouse adipocytes and myotubes. The results show that chronic arsenic exposure significantly decreased insulin-stimulated glucose uptake (ISGU) in correlation with reduced expression of insulin-regulated glucose transporter type 4 (Glut4). Expression of Sirt3, a mitochondrial deacetylase, was dramatically decreased along with its associated transcription factor, forkhead box O3 (FOXO3a) upon arsenic exposure. A decrease in mitochondrial membrane potential (Δψm) was observed in both 3T3L1 adipocytes and C2C12 myotubes treated by arsenic. Reduced FOXO3a activity by arsenic exhibited a decreased binding affinity to the promoters of both manganese superoxide dismutase (MnSOD) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, a broad and powerful regulator of reactive oxygen species (ROS) metabolism. Forced expression of Sirt3 or MnSOD in mouse myotubes elevated Δψm and restored ISGU inhibited by arsenic exposure. Our results suggest that Sirt3/FOXO3a/MnSOD signaling plays a significant role in the inhibition of ISGU induced by chronic arsenic exposure.
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http://dx.doi.org/10.1093/toxsci/kfv089DOI Listing
August 2015

Epigenetic targets of arsenic: emphasis on epigenetic modifications during carcinogenesis.

J Environ Pathol Toxicol Oncol 2015 ;34(1):63-84

Center for Research on Environmental Disease and Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky.

DNA methylation and histone modification promote opening and closure of chromatin structure, which affects gene expression without altering the DNA sequence. Epigenetic markers regulate the dynamic nature of chromatin structure at different levels: DNA, histone, noncoding RNAs, as well as the higher-order chromatin structure. Accumulating evidence strongly suggests that arsenic-induced carcinogenesis involves frequent changes in the epigenetic marker. However, progress in identifying arsenic-induced epigenetic changes has already been made using genome-wide approaches; the biological significance of these epigenetic changes remains unknown. Moreover, arsenic-induced changes in the chromatin state alter gene expression through the epigenetic mechanism. The current review provides a summary of recent literature regarding epigenetic changes caused by arsenic in carcinogenesis. We highlight the transgenerational studies needed to explicate the biological significance and toxicity of arsenic over a broad spectrum.
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http://dx.doi.org/10.1615/jenvironpatholtoxicoloncol.2014012066DOI Listing
July 2015

Blackberry extract inhibits UVB-induced oxidative damage and inflammation through MAP kinases and NF-κB signaling pathways in SKH-1 mice skin.

Toxicol Appl Pharmacol 2015 Apr 11;284(1):92-99. Epub 2015 Feb 11.

Department of Toxicology and Cancer Biology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA.

Extensive exposure of solar ultraviolet-B (UVB) radiation to skin induces oxidative stress and inflammation that play a crucial role in the induction of skin cancer. Photochemoprevention with natural products represents a simple but very effective strategy for the management of cutaneous neoplasia. In this study, we investigated whether blackberry extract (BBE) reduces chronic inflammatory responses induced by UVB irradiation in SKH-1 hairless mice skin. Mice were exposed to UVB radiation (100 mJ/cm(2)) on alternate days for 10 weeks, and BBE (10% and 20%) was applied topically a day before UVB exposure. Our results show that BBE suppressed UVB-induced hyperplasia and reduced infiltration of inflammatory cells in the SKH-1 hairless mice skin. BBE treatment reduced glutathione (GSH) depletion, lipid peroxidation (LPO), and myeloperoxidase (MPO) in mouse skin by chronic UVB exposure. BBE significantly decreased the level of pro-inflammatory cytokines IL-6 and TNF-α in UVB-exposed skin. Likewise, UVB-induced inflammatory responses were diminished by BBE as observed by a remarkable reduction in the levels of phosphorylated MAP Kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, BBE also reduced inflammatory mediators such as cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) levels in UVB-exposed skin. Treatment with BBE inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mouse skin. Immunohistochemistry analysis revealed that topical application of BBE inhibited the expression of 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG), cyclobutane pyrimidine dimers (CPD), proliferating cell nuclear antigen (PCNA), and cyclin D1 in UVB-exposed skin. Collectively, these data indicate that BBE protects from UVB-induced oxidative damage and inflammation by modulating MAP kinase and NF-κB signaling pathways.
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http://dx.doi.org/10.1016/j.taap.2015.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374016PMC
April 2015

Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways.

Toxicol Appl Pharmacol 2014 Dec 23;281(2):230-41. Epub 2014 Oct 23.

Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA; Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA. Electronic address:

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5μM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis.
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http://dx.doi.org/10.1016/j.taap.2014.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433031PMC
December 2014

Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin.

Toxicol Appl Pharmacol 2014 Oct 22;280(1):127-37. Epub 2014 Jul 22.

Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA; Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USA. Electronic address:

Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE2 and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2'-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways.
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http://dx.doi.org/10.1016/j.taap.2014.06.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330564PMC
October 2014

Luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

PLoS One 2012 31;7(12):e52279. Epub 2012 Dec 31.

Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America.

Angiogenesis, the formation of new blood vessels from pre-existing vascular beds, is essential for tumor growth, invasion, and metastasis. Luteolin is a common dietary flavonoid found in fruits and vegetables. We studied the antiangiogenic activity of luteolin using in vitro, ex vivo, and in vivo models. In vitro studies using rat aortic ring assay showed that luteolin at non-toxic concentrations significantly inhibited microvessel sprouting and proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Luteolin also inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Gelatin zymographic analysis demonstrated the inhibitory effect of luteolin on the activation of matrix metalloproteinases MMP-2 and MMP-9. Western blot analysis showed that luteolin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 in HUVECs. Proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α level were significantly reduced by the treatment of luteolin in PC-3 cells. Luteolin (10 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that luteolin inhibited tumorigenesis by targeting angiogenesis. CD31 and CD34 immunohistochemical staining further revealed that the microvessel density could be remarkably suppressed by luteolin. Moreover, luteolin reduced cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, ERK, mTOR, P70S6K, MMP-2, and MMP-9 expressions. Taken together, our findings demonstrate that luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052279PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3534088PMC
July 2013