Pranav Gupta

Expression and clinical significance of CXCR5/CXCL13 in human non‑small cell lung carcinoma

CXCR5 and/or CXCL13 expression is elevated in certain carcinomas and lymphomas. To determine if these factors are involved in progression of non-small cell lung cancer (LuCa), we evaluated their expression in patients with various forms of this disease. Lung biopsies from patients with non-neoplastic cells (n=8), squamous cell carcinoma (SCC; n=24), or adenocarcinoma (AC; n=54) were stained for CXCR5. Histopathological analysis of these samples showed significantly higher expression of CXCR5 (p<0.001) in carcinomas (i.e., SCCs and ACs) relative to non-neoplastic lung tissue. Nuclear and membrane CXCR5 intensities were highest in ACs, with median values of 185 and 130, respectively, followed by SCCs with median values of 170 and 110, respectively. The lowest nuclear and membrane expressions of CXCR5 were found in non-neoplastic tissues, having median values of 142 and 90, respectively. Sera from SCC patients (n=17), AC patients (n=14), and healthy controls (n=9) were tested for the presence of CXCL13. Serum CXCL13 levels in LuCa patients were higher than in healthy controls. CXCR5 expression in cell lines of human non-small cell lung carcinoma (NCI-H1915) and small cell lung carcinoma (SW-1271) were evaluated by flow cytometry. CXCR5 expression was higher in NCI-H1915 cells relative to SW-1271 cells. The functional significance of CXCR5 expression was tested in a migration assay. In response to CXCL13, more NCI-H1915 cells migrated than SW-1271 cells. These findings suggest that the CXCR5-CXCL13 axis influences LuCa progression. After validation in larger patient groups, CXCR5 and CXCL13 may prove useful as biomarkers for LuCa. Correspondingly, blockade of this axis could serve as an effective therapy for LuCa.

A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo

ATP-binding cassette subfamily G member 2 (ABCG2) is a member of the ABC transporter superfamily proteins, which has been implicated in the development of multidrug resistance (MDR) in cancer, apart from its physiological role to remove toxic substances out of the cells. The diverse range of substrates of ABCG2 includes many antineoplastic agents such as topotecan, doxorubicin and mitoxantrone. ABCG2 expression has been reported to be significantly increased in some solid tumors and hematologic malignancies, correlated to poor clinical outcomes. In addition, ABCG2 expression is a distinguishing feature of cancer stem cells, whereby this membrane transporter facilitates resistance to the chemotherapeutic drugs. To enhance the chemosensitivity of cancer cells, attention has been focused on MDR modulators. In this study, we investigated the effect of a tetrodotoxin-resistant sodium channel blocker, A-803467 on ABCG2-overexpressing drug selected and transfected cell lines. We found that at non-toxic concentrations, A-803467 could significantly increase the cellular sensitivity to ABCG2 substrates in drug-resistant cells overexpressing either wild-type or mutant ABCG2. Mechanistic studies demonstrated that A-803467 (7.5 μM) significantly increased the intracellular accumulation of [3H]-mitoxantrone by inhibiting the transport activity of ABCG2, without altering its expression levels. In addition, A-803467 stimulated the ATPase activity in membranes overexpressed with ABCG2. In a murine model system, combination treatment of A-803467 (35 mg/kg) and topotecan (3 mg/kg) significantly inhibited the tumor growth in mice xenografted with ABCG2-overexpressing cancer cells. Our findings indicate that a combination of A-803467 and ABCG2 substrates may potentially be a novel therapeutic treatment in ABCG2-positive drug resistant cancers.

Prediction and characterization of helper T‐cell epitopes from pneumococcal surface adhesin A

Pneumococcal surface adhesin A (PsaA) is a multifunctional lipoprotein known to bind nasopharyngeal epithelial cells, and is significantly involved in bacterial adherence and virulence. Identification of PsaA peptides that optimally bind human leucocyte antigen (HLA) and elicit a potent immune response would be of great importance to vaccine development. However, this is hindered by the multitude of HLA polymorphisms in humans. To identify the conserved immunodominant epitopes, we used an experimental dataset of 28 PsaA synthetic peptides and in silico methods to predict specific peptide‐binding to HLA and murine MHC class II molecules. We also characterized spleen and cervical lymph node (CLN) ‐derived T helper (Th) lymphocyte cytokine responses to these peptides after Streptococcus pneumoniae strain EF3030 challenge in mice. Individual, yet overlapping, peptides 15 amino acids in length revealed residues of PsaA that consistently caused the highest interferon‐γ, interleukin‐2 (IL‐2), IL‐5 and IL‐17 responses and proliferation as well as moderate IL‐10 and IL‐4 responses by ex vivo re‐stimulated splenic and CLN CD4+ T cells isolated from S. pneumoniae strain EF3030‐challenged F1 (B6 × BALB/c) mice. In silico analysis revealed that peptides from PsaA may interact with a broad range of HLA‐DP, ‐DQ and ‐DR alleles, due in part to regions lacking β‐turns and asparagine endopeptidase sites. These data suggest that Th cell peptides (7, 19, 20, 22, 23 and 24) screened for secondary structures and MHC class II peptide‐binding affinities can elicit T helper cytokine and proliferative responses to PsaA peptides.

Effect of Y 6 , an epigallocatechin gallate derivative, on reversing doxorubicin drug resistance in human hepatocellular carcinoma cells

Cancer cells can acquire resistance to a wide variety of diverse and unrelated drugs, this phenomenon is termed multidrug resistance (MDR). Multidrug resistance has been an obstacle to the success of cancer chemotherapy. The present study investigated the reversal effect of Y6, a new compound obtained by chemically modifying the structure of epigallocatechin-3-gallate (EGCG) extracted from green tea. Y6 was proven to be effective in inhibiting cell proliferation and reversing drug resistance in doxorubicin (DOX) resistant human hepatocellular carcinoma cells (BEL-7404/DOX). BEL-7404/DOX cells were treated with either doxorubicin combination regimen (doxorubicin plus Y6 or epigallocatechin-3-gallate or verapamil separately) or doxorubicin alone. The results showed that cell proliferation was inhibited and late cell apoptosis increased in the combination treatment group, especially in the group treated with doxorubicin plus Y6. Further analysis revealed that the expressions of hypoxia-inducible factor-1α and multidrug resistance 1/P-glycoprotein decreased at both messenger RNA and protein levels by treatments with combined drugs compared to doxorubicin alone. Our results indicated that Y6, as a drug resistance reversal agent, increased the sensitivity of drug resistant cells to doxorubicin. The mechanisms of actions of Y6 in reversal effect were associated with the decreased expression of hypoxia-inducible factor-1α and multidrug resistance 1/P-glycoprotein.

Abstract 2128: The effects of Quercetin on prostate cancer

Prostate cancer (PCa) affects nearly 80% of men worldwide and is the second leading killer after lung cancer. The efficacy of current treatments offered in the clinics is highly compromised due to indolent nature of PCa, which provides large window of opportunity for prevention. Hence, the major focus of this study is to determine the chemo-preventive effects of Quercetin, a bioflavonoid, on prostate cancer. Effect of Quercetin on cell viability and IC50 was determined by MTT assay. The effect of Quercetin on cell motility was determined by wound healing assay. Potential role of Quercetin on cell cycle, apoptosis as well as genes involved in cell motility and invasion was determined using flow cytometry, Real-time qPCR and ELISA. Furthermore, Quercetin induced changes in signaling molecules involved in cell survival/apoptosis, cell cycle and cytoskeletal rearrangement was determined using antibody microarray. Prostate cancer cells treated with Quercetin showed dose and time dependent inhibition of proliferation/viability, and induction of apoptosis as compared to normal prostatic epithelial cells and untreated controls. Prostate cancer cell motility was inhibited in Quercetin treated cells. Prostate cancer cells were arrested in G2 phase of the cell cycle following Quercetin treatment. In addition to these, we found differential expression of caspases, matrix metalloproteinases (MMPs) and tissue inhibitor of MMPs in different PCa cell lines compared to untreated controls. Furthermore, antibody microarray analysis demonstrated selective modulation of genes and associated signaling cascades responsible for apoptosis induction, cellular motility, adhesion and invasion in Quercetin treated cells compared to controls. These findings suggest Quercetin as a potent chemo-preventive agent. In addition to this it can be also used with chemotherapeutic agents directed to G2 phase of the cell cycle, which may improve the efficacy of chemotherapeutics offered in clinics to treat advance prostate cancer. Citation Format: Ashley B. Ward, Pranav Gupta, Gurpreet Kaur, Hina Mir, James W. Lillard, Shailesh Singh. The effects of Quercetin on prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2128. doi:10.1158/1538-7445.AM2014-2128

Abstract 4003: Clinical and biological significance of CXCR6 in lung cancer

Lung cancer is the leading cause of cancer related deaths worldwide in both men and women. Metastasis is responsible for most lung cancer related deaths; therefore, a better understanding of the metastatic processes and therapies designed to prevent the spread of cancer cells are greatly needed. The specific mechanisms that promote metastases have not been fully elucidated. Among all known chemokine receptors involved in cancer progression, most cancer cells including lung cancer express CXCR4. Furthermore, involvement of CXCR4 and CCR7 in breast cancer and CXCR4, CCR9 and CX3CR1 in prostate cancer progression further indicates that multiple chemokine receptors are involved in dictating the multi-step process of metastasis. In this study, we show significantly higher expression of CXCR6 and CXCL16 in lung cancer tissues compared to normal matched tissues. Expression of CXCR6 was significantly higher in adeno- carcinoma compared to squamous cell carcinoma. It addition to these, serum levels of CXCL16, the only natural ligand for CXCR6, was also significantly higher in lung cancer patients compared to normal healthy donor. Furthermore, serum CXCL16 was also significantly higher in patients with adenocarcinoma compared to patients with squamous cell carcinoma. Impact of this chemokines-receptor axis was determined in lung cancer cell lines in vitro, which express CXCR6 and CXCL16. Like tissues, lung cancer cells showed higher expression of CXCR6 and CXCL16 compared to normal lung epithelial cells (NuLi-1). Interestingly, expression of CXCR6 was highest in cell lines derived form adenocarcinoma followed by cell lines from squamous cell carcinoma. Furthermore, we demonstrate that CXCR6 and soluble CXCL16 interaction plays a crucial role in lung cancer cells migration and invasion. The mechanism underlying these clinically and biologically important findings need to be further explored. Increased serum CXCL16 in lung cancer patients with metastatic disease required further validation as a potential therapeutic target and/or diagnostic marker for lung cancer. Citation Format: Hina Mir, Pranav Gupta, Rajesh Singh, Praveen K. Sharma, Gurpreet Kaur, Ashley B. Ward, William E. Grizzle, James W. Lillard, Shailesh Singh. Clinical and biological significance of CXCR6 in lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4003. doi:10.1158/1538-7445.AM2014-4003

Selonsertib (GS-4997), an ASK1 inhibitor, antagonizes multidrug resistance in ABCB1- and ABCG2-overexpressing cancer cells

Overexpression of ATP-binding cassette (ABC) transporters is one of the most important mechanisms responsible for the development of multidrug resistance (MDR). Selonsertib, a serine/threonine kinase inhibitor, targets apoptosis signal-regulating kinase 1 (ASK1) and is now in phase III clinical trial for the treatment of non-alcoholic steatohepatitis (NASH). In this study, we investigated whether selonsertib could reverse MDR-mediated by ABC transporters, including ABCB1, ABCG2, ABCC1 and ABCC10. The results showed that selonsertib significantly reversed ABCB1- and ABCG2-mediated MDR, but not MDR-mediated by ABCC1 or ABCC10. Mechanism studies indicated that the reversal effect of selonsertib was related to the attenuation of the efflux activity of ABCB1 and ABCG2 transporters, without the protein level decrease or change in the subcellular localization of ABCB1 or ABCG2. Selonsertib stimulated the ATPase activity of ABCB1 and ABCG2 in a concentration-dependent manner, and in silico docking study showed selonsertib could interact with the substrate-binding sites of both ABCB1 and ABCG2. This study provides a clue into a novel treatment strategy, which includes a combination of selonsertib with antineoplastic drugs to attenuate MDR-mediated by ABCB1 or ABCG2 in cancer cells overexpressing these transporters.

Icotinib improves progression free survival in epidermal growth factor receptor positive non-small cell lung cancer patients

Lung cancer, as defined by the National Cancer Institute, is the uncontrolled proliferation of cells lining the air passages and tissues of the lungs (1). Lung cancer is the second most common cancer in both men and women (with prostate and breast as the most common in men and women, respectively).

The epigallocatechin gallate derivative Y6 reverses drug resistance mediated by the ABCB1 transporter both in vitro and in vivo

Previously, we reported that Y6, a new epigallocatechin gallate derivative, is efficacious in reversing doxorubicin (DOX)--mediated resistance in hepatocellular carcinoma BEL-7404/DOX cells. In this study, we evaluated the efficacy of Y6 in reversing drug resistance both in vitro and in vivo by determining its effect on the adenosine triphosphate-binding cassette protein B1 transporter (ABCB1 or P-glycoprotein, P-gp). Our results showed that Y6 significantly sensitized cells overexpressing the ABCB1 transporter to anticancer drugs that are ABCB1 substrates. Y6 significantly stimulated the adenosine triphosphatase activity of ABCB1. Furthermore, Y6 exhibited a higher docking score as compared with epigallocatechin gallate inside the transmembrane domain of ABCB1. In addition, in the nude mouse tumor xenograft model, Y6 (110 mg/kg, intragastric administration), in combination with doxorubicin (2 mg/kg, intraperitoneal injection), significantly inhibited the growth of BEL-7404/DOX cell xenograft tumors, compared to equivalent epigallocatechin gallate. In conclusion, Y6 significantly reversed ABCB1-mediated multidrug resistance and its mechanisms of action may result from its competitive inhibition of the ABCB1 drug efflux function.

Abstract 4158: Targeting the imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia through a novel BCR-ABL inhibitor

Chronic Myeloid Leukemia (CML), a hematological malignancy, is largely caused by the Philadelphia (Ph) chromosome carrying the Break point Cluster Region-Abelson (BCR-ABL) oncogene. Imatinib is a BCR-ABL-targeted therapy and considered the standard of care in CML management. Resistance to imatinib therapy often develops because of mutations in the BCR-ABL kinase domain. In this study, we evaluated PBA2, a novel BCR-ABL inhibitor, for its anti-cancer activity against BCR-ABL expressing BaF3 cells. PBA2 shows potent activity against wild-type and T315I mutated BaF3 cells as compared with imatinib. PBA2 inhibited the phosphorylation of BCR-ABL and its downstream signaling in BaF3/WT and BaF3/T315I cells. PBA2 inhibited the mRNA expression of BCR-ABL in BaF3/WT and BaF3/T315I cells. Mechanistically, PBA2 increased the cell population in sub G1 phase of the cell cycle, induced apoptosis and elevated ROS production in both BaF3/WT and BaF3/T315I cells. Taken together, our results indicate that PBA2 exhibits anti-proliferative effects and inhibits the imatinib-resistant T315I BCR-ABL mutation. PBA2 may be a novel drug candidate for overcoming the resistance to imatinib in CML patients. Note: This abstract was not presented at the meeting. Citation Format: Pranav Gupta, Rishil Kathawala, Liuya Wei, Fang Wang, XiaoKun Wang, Brian J. Druker, Li-Wu FU, Zhe-Sheng Chen. Targeting the imatinib-resistant BCR-ABL T315I mutation in chronic myeloid leukemia through a novel BCR-ABL inhibitor [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 4158. doi:10.1158/1538-7445.AM2017-4158