Neeraj Kapur

CXCR6-CXCL16 axis promotes prostate cancer by mediating cytoskeleton rearrangement via Ezrin activation and α v β 3 integrin clustering

Cytoskeletal rearrangement is required for migration and invasion, which are the key steps of cancer metastasis. Ezrin and integrin co-ordinate these processes by regulating cellular adhesion and cytoskeletal polymerization-depolymerization. It is also well established that chemokine-chemokine receptor axis plays a crucial role in regulating cancer cell migration and invasion. In this study, we show involvement of CXC chemokine receptor 6 (CXCR6) and its only natural ligand CXCL16 in pathobiology of prostate cancer (PCa). CXCR6 is highly expressed in PCa tissues and cell lines (LNCaP and PC3), relative to normal tissue and cells. CXCR6 expression in PCa tissues correlated with higher Gleason score. Similarly, aggressive PCa cells (PC3) show high CXCR6 compared to less aggressive LNCaP. Besides, PC3 cells show higher MMPs expression compared to LNCaP cells following CXCL16 stimulation. Intriguingly, CXCR6-CXCL16 interaction in PCa cells promotes Ezrin activation, αvβ3 integrin clustering and capping at the leading edge in FAK/PI3K/PKC dependent manner, thereby modifying cellular adhesion as well as motility. Together these results demonstrate that CXCL16 stimulation changes cytoskeletal dynamics resulting in enhanced migration, invasion and adhesion to endothelial cells, ultimately enabling PCa cells to achieve their metastatic goal.

Andrographolide inhibits prostate cancer by targeting cell cycle regulators, CXCR3 and CXCR7 chemokine receptors.

ABSTRACT Despite state of the art cancer diagnostics and therapies offered in clinic, prostate cancer (PCa) remains the second leading cause of cancer-related deaths. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of Andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on cell cycle phases in LNCaP, C4-2b and PC3 cells compared to retinoblastoma protein (RB−/−) and CDKN2A lacking DU-145 cells. G2/M transition was blocked in LNCaP, C4-2b and PC3 after AG treatment whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to differential activation of cell cycle regulators in these cell lines. Levels of cyclin A2 after AG treatment increased in all PCa cells line. Cyclin B1 levels increased in LNCaP and PC3, decreased in C4-2b and showed no difference in DU-145 cells after AG treatment. AG decreased cyclin E2 levels only in PC3 and DU-145 cells. It also altered Rb, H3, Wee1 and CDC2 phosphorylation in PCa cells. Intriguingly, AG reduced cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggests its potential use as a therapeutic and/or preventive agent for PCa.

CCR6 expression in colon cancer is associated with advanced disease and supports epithelial-to-mesenchymal transition

Background:Adjuvant chemotherapy offered to treat colon cancer is based on the TNM staging system, which often fails due to molecular heterogeneity and undefined molecular mechanisms independent of TNM. Therefore, identification of markers to better predict therapeutic option and outcome is needed. In this study we have characterised the clinical association of CCR6 with colon cancer and defined CCR6-mediated molecular pathway.Methods:Immunohistochemistry, RT-qPCR, western blot and FACS were used to determine expression of CCR6 and/or EMT markers in colon tissues/cells. BrdU assay and trans-well system were used to determine cell proliferation, migration and invasion in response to CCL20.Results:CCR6 was higher in cancer cases compared to normal adjacent tissue and expression was associated with nodal status and distant metastasis. Similarly, CCR6 expression was higher in cells derived from node-positive cases and highest expression was in cells derived from metastatic cases. Significant changes in EMT markers, that is, E-cadherin, vimentin, β-catenin, N-cadherin, α-SMA, SNAILl and ZEB1 were observed in response to CCL20 along with decreased proliferation, increased migratory and invasive potential.Conclusions:Results suggest CCR6 as a potential therapeutic target as well as biomarker in addition to nodal status for predicting therapeutic option.

Quercetin inhibits prostate cancer by attenuating cell survival and inhibiting anti-apoptotic pathways

BackgroundDespite recent advances in diagnosis and treatment, prostate cancer (PCa) remains the leading cause of cancer-related deaths in men. Current treatments offered in the clinics are often toxic and have severe side effects. Hence, to treat and manage PCa, new agents with fewer side effects or having potential to reduce side effects of conventional therapy are needed. In this study, we show anti-cancer effects of quercetin, an abundant bioflavonoid commonly used to treat prostatitis, and defined quercetin-induced cellular and molecular changes leading to PCa cell death.MethodsCell viability was assessed using MTT. Cell death mode, mitochondrial outer membrane potential, and oxidative stress levels were determined by flow cytometry using Annexin V-7 AAD dual staining kit, JC-1 dye, and ROS detection kit, respectively. Antibody microarray and western blot were used to delineate the molecular changes induced by quercetin.ResultsPCa cells treated with various concentrations of quercetin showed time- and dose-dependent decrease in cell viability compared to controls, without affecting normal prostate epithelial cells. Quercetin led to apoptotic and necrotic cell death in PCa cells by affecting the mitochondrial integrity and disturbing the ROS homeostasis depending upon the genetic makeup and oxidative status of the cells. LNCaP and PC-3 cells that have an oxidative cellular environment showed ROS quenching after quercetin treatment while DU-145 showed rise in ROS levels despite having a highly reductive environment. Opposing effects of quercetin were also observed on the pro-survival pathways of PCa cells. PCa cells with mutated p53 (DU-145) and increased ROS showed significant reduction in the activation of pro-survival Akt pathway while Raf/MEK were activated in response to quercetin. PC-3 cells lacking p53 and PTEN with reduced ROS levels showed significant activation of Akt and NF-κB pathway. Although some of these changes are commonly associated with oncogenic response, the cumulative effect of these alterations is PCa cell death.ConclusionsOur results demonstrated quercetin exerts its anti-cancer effects by modulating ROS, Akt, and NF-κB pathways. Quercetin could be used as a chemopreventive option as well as in combination with chemotherapeutic drugs to improve clinical outcomes of PCa patients.

Abstract 4642: Quercetin inhibits prostate cancer by modulating molecules involved in apoptosis and cell proliferation

Prostate cancer (PCa) remains the second leading cause of death in men, despite the multimodal options offered in the clinics. Long latency and indolent nature of PCa provides window of opportunity for preventive interventions using natural and synthetic agents. Quercetin, a bioflavonoid abundant in fruits and vegetables, has been reported to inhibit growth in cancer and reduce inflammation in conditions like prostatitis. Considering this, we investigated the chemo-preventive effects of Quercetin on PCa. The effects of Quercetin on apoptosis and associated proteins on both RNA and protein levels were determined using apoptosis assay, real-time qPCR and western blot, respectively. Furthermore, an antibody microarray was performed to observe changes induced by Quercetin on signaling molecules and possible mechanism of cell survival/apoptosis in PCa. PCa cells treated with Quercetin showed decreased cell proliferation in a time and dose dependent manner, while no significant effect was seen on normal prostate cells. Quercetin treatment also significantly induced apoptosis in PCa cells, as compared with untreated controls. Different phosphorylation status of key molecules (Bcl-2, Akt-1 and NF-k B) was observed following Quercetin treatment in PCa cell lines, as compared with untreated controls, which are known to be involved in supporting PCa progression. These results suggest that Quercetin could be a potential chemo-preventative agent capable of slowing down the progression of PCa, by increasing apoptosis and decreasing cell proliferation. Note: This abstract was not presented at the meeting. Citation Format: Ashely B. Ward, Hina Mir, Neeraj Kapur, Shailesh Singh. Quercetin inhibits prostate cancer by modulating molecules involved in apoptosis and cell proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4642. doi:10.1158/1538-7445.AM2015-4642

Cinnamtannin B-1 inhibits cell survival molecules and induces apoptosis in colon cancer

Colon cancer patients receiving chemotherapy continue to be burdened with therapeutic failure and adverse side effects, yielding a need to develop more effective treatments. The present study investigates Cinnamtannin B-1 (CTB-1) as a potential low-toxicity therapeutic alternative for colon cancer. CTB-1-treated DLD-1, COLO 201 and HCT-116 (WT p53 and p53 null) colon cancer cells and CCD 841 CoN normal colon epithelial cells were assessed for changes in survival using MTT assay. The effects of CTB-1 on cell cycle progression and the apoptosis of colon cancer cells were measured using flow cytometry and/or immunofluorescence. The expression profiles of cell survival molecules, particularly apoptotic proteins, in the colon cancer cells were evaluated following CTB-1 treatment via antibody array, then validated by western blot analysis. Additionally, the potential synergy between CTB-1 and 5-fluorouracil (5-FU), a conventional chemotherapeutic agent used in the treatment of colon cancer, against colon cancer cells was assessed using MTT assay and Calcusyn software. The results revealed that CTB-1 signifi-cantly decreased the survival of the DLD-1, COLO 201 and HCT-116 cells in a time and/or dose-dependent manner, with minimal cytotoxicity to normal colon cells. CTB-1 treatment was shown to induce cell cycle arrest and apoptosis of DLD-1 and COLO 201 cells. Of note, CTB-1 modulated the expression of several cell survival molecules, which tend to be deregulated in colon cancer, including p53, a key transcription factor involved in apoptosis. The downstream regulation of Bcl-2 and Bak expression, as well as cytochrome c release into the cytosol, was also observed following CTB-1 treatment. Furthermore, CTB-1 was shown to significantly enhance the potency of 5-FU via a synergistic drug interaction. This study reveals for the first time, to the best of our knowledge, the ability of CTB-1 to decrease the survival of colon cancer cells through pro-apoptotic mechanisms and display synergy with conventional chemotherapy, demonstrating the potential therapeutic benefit of CTB-1 in colon cancer.

Abstract 5252: Quercetin inhibits prostate cancer by modulating ROS and key regulators of apoptosis and cell survival

Long latency and indolent nature of prostate cancer (PCa) provides a window of opportunity for preventive interventions using natural and synthetic agents. Hence, the focus of this study was to ascertain the chemopreventive role of Quercetin, a bioflavonoid, commonly used to treat prostatitis. Human PCa cells (LNCaP, DU145 and PC3) were treated with different concentrations of Quercetin and its effect on cell survival and apoptosis was determined by MTT assay. Human PCa cells treated with Quercetin showed significant reduction in cell viability and proliferation compared with untreated controls, which was dose and time dependent. In addition to this our FACS analysis showed higher percentage of apoptotic cells after Quercetin treatment compared to untreated cells. Quercetin induced apoptosis in PCa cells is a cumulative effect of modulation of key apoptotic proteins, changes in mitochondrial membrane potential and ROS production. Our results demonstrate that Quercetin is a potent chemopreventive agent, which may improve outcomes of PCa by inhibiting mechanisms involved in tumor progression. Citation Format: Ashley B. Ward, Hina Mir, Neeraj Kapur, Guru Sonpavde, Shailesh Singh. Quercetin inhibits prostate cancer by modulating ROS and key regulators of apoptosis and cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5252. doi:10.1158/1538-7445.AM2017-5252

Abstract 312: Anti-proliferative effects of cinnamon extract in colon cancer

Colon cancer is the third most lethal cancer worldwide, and is estimated to cause 49,190 cancer-related deaths in the U.S. in 2016 alone. Current treatments for colon cancer are limited in their capacity to target tumor cells employing adaptive mechanisms necessary for survival and proliferation, often resulting in poor prognosis. Therefore, it is critical to develop novel target-specific therapeutic approaches with minimal cytotoxicity to normal cells, in order to mitigate potential side effects. Proanthocyanidins represent a subclass of flavonoids, which have been widely investigated for their chemopreventive and therapeutic potential. Cinnamtannin B-1 (CTB-1) is a proanthocyanidin shown to have anti-cancer effects in several cancers, but its anti-cancer potential in colon cancer has not been tested. Hence, the focus of this study was to determine the anticancer effects of CTB-1 in colon cancer. Our results show that CTB-1 treatment significantly decreases the cell viability and proliferation of DLD-1 and COLO 201 human colon cancer cells in a time and dose-dependent manner. In addition, CTB-1 treatment modulated the cell cycle progression of colon cancer cells via G2/M arrest, coupled with a reduction of cells in the S phase. The expression and/or phosphorylation of key molecules involved in apoptosis and cell cycle progression were also modulated in response to CTB-1 treatment. Collectively, these findings, for the first time, shed light on the anti-proliferative effects of CTB-1 in colon cancer, and further demonstrate its potential as a novel therapeutic/preventive agent. Citation Format: Patrick P. Carriere, Hina Mir, Neeraj Kapur, Clarence E. Clark, Shailesh Singh. Anti-proliferative effects of cinnamon extract in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 312. doi:10.1158/1538-7445.AM2017-312

Abstract 5254: Andrographolide prevents prostate cancer by targeting CXCR3/CXCR7 and regulators of cell cycle

Despite state of the art cancer diagnostics and therapies offered in clinics, prostate cancer (PCa) remains the second leading cause of cancer-related deaths in men. Hence, more robust therapeutic/preventive regimes are required to combat this lethal disease. In the current study, we have tested the efficacy of andrographolide (AG), a bioactive diterpenoid isolated from Andrographis paniculata, against PCa. This natural agent selectively affects PCa cell viability in a dose and time-dependent manner, without affecting primary prostate epithelial cells. Furthermore, AG showed differential effect on phases of cell cycle in LNCaP, C4-2b and PC3 cell lines compared to retinoblastoma protein (RB-/-) lacking DU-145 cells. This agent blocked the G2/M transition in LNCaP, C4-2b and PC3 whereas DU-145 cells failed to transit G1/S phase. This difference was primarily due to switching of the activation states of cell cycle regulators in these cell lines. AG induced its effect mainly via cyclin A2 and B1 in LNCaP, C4-2b and PC3 cell lines; whereas, cyclin E related regulation was affected in DU-145 cells. Phosphorylation status of Wee1, a nuclear kinase belonging to the Ser/Thr family and CDC2 was also affected by AG. Intriguingly, AG affected cell viability and the ability of PCa cells to migrate via modulating CXCL11 and CXCR3 and CXCR7 expression. The significant impact of AG on cellular and molecular processes involved in PCa progression suggest this agent has potential to be used as a therapeutic and/or preventive modality. Citation Format: Hina Mir, Neeraj Kapur, Rajesh Singh, Guru Sonpavde, James W. Lillard, Shailesh Singh. Andrographolide prevents prostate cancer by targeting CXCR3/CXCR7 and regulators of cell cycle. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5254.

Abstract 1701: CCR6 associates with colon cancer metastasis

Despite established benefits of screening, colon cancer remains a leading cause of cancer death in the U.S. Majority of colon cancer deaths result from metastasis. Effective treatments are not available for advanced disease because molecular mechanisms of initiation and progression of this disease are yet to be defined. Chemokine-chemokine receptor interaction plays an important role in cancer progression. In this study, using colon cancer tissue microarray, we have shown that expression of CCR6 was significantly higher in advanced colon cancer (p Citation Format: Neeraj Kapur, Hina Mir, Clarence E. Clark, Uma Krishnamurti, Derrick J. Beech, James W. Lillard, Shailesh Singh. CCR6 associates with colon cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1701.