Sheefa Mirza

Current Drugs and Drug Targets in Non-Small Cell Lung Cancer: Limitations and Opportunities

Lung cancer is a serious health problem and leading cause of death worldwide due to its high incidence and mortality. More than 80% of lung cancers feature a non-small cell histology. Over few decades, systemic chemotherapy and surgery are the only treatment options in this type of tumor but due to their limited efficacy and overall poor survival of patients, there is an urge to develop newer therapeutic strategies which circumvent the problems. Enhanced knowledge of translational science and molecular biology have revealed that lung tumors carry diverse driver gene mutations and adopt different intracellular pathways leading to carcinogenesis. Hence, the development of targeted agents against molecular subgroups harboring critical mutations is an attractive approach for therapeutic treatment. Targeted therapies are clearly more preferred nowadays over systemic therapies because they target tumor specific molecules resulting with enhanced activity and reduced toxicity to normal tissues. Thus, this review encompasses comprehensive updates on targeted therapies for the driver mutations in non-small cell lung cancer (NSCLC) and the potential challenges of acquired drug resistance faced in the field of targeted therapy along with the imminent newer treatment modalities against lung cancer.

Enhancement of the cytotoxic effects of Cytarabine in synergism with Hesperidine and Silibinin in Acute Myeloid Leukemia: An in-vitro approach

OBJECTIVES Acute Myeloid Leukemia (AML) therapy continues to be a daunting challenge. Cytosine Arabinoside (Ara-C) is widely used to treat hematological malignancy in humans, but often becomes ineffective because of increased resistance to the drug which may lead to a worse prognosis. Therefore new strategies are needed to understand the mechanism responsible for drug resistance and to develop new therapies to overcome it. Research evidence based on natural compounds used alone or in combination with current chemotherapeutic agents proved their efficacy to treat and prevent cancer. Hesperidin and Silibinin displayed anti-cancer activity against various types of cancers and cell lines and can be used in combination with Cytarabine with the aim to increase cytotoxicy profile and reduction in drug resistance. Experimental Work: Primary cells obtained from AML patients bone marrow were used to develop in-vitro model and further exposed to various concentration of Cytarabine (10 nM-5000 nM), Hesperidin (0.5 μM-100 μM) and Silibinin (0.5 μM-100 μM) alone and in combination with Cytarabine (Hesperidin-25 μM, Silibinin10 μM) to check cytotoxicity using MTT assay. Synergistic effect was evaluated by Combination Index method. RESULT AND CONCLUSION In-vitro study of Hesperidin and Silibinin indicated their cytotoxicity at IC 50 value 50.12 μM and 16.2 μM, respectively. Combination Index study revealed Hesperidin and Silibinin both showed synergistic potential and decreased the IC 50 value of Cytarabine by ~5.9 and ~4.5 folds, respectively. Both natural compounds showed potential anti-leukemic activity hence may be used for AML therapy alone or in combination with other chemotherapeutic agents.

Synergism of Curcumin and Cytarabine in the Down Regulation of Multi-Drug Resistance Genes in Acute Myeloid Leukemia.

BACKGROUND The aim of the study was to find a role of Curcumin from natural source to overcome drug resistance as well as to reduce cytotoxicity profile of the drug in Acute Myeloid Leukemia patients. MATERIAL AND METHODS Primary leukemic cells were obtained from AML patients bone marrow. These cells were then exposed to different concentration of cytarabine and curcumin to find out IC50 values and also its effect on MDR genes like MDR1, BCRP, LRP and FLT3 by RT-PCR method. RESULT & CONCLUSION Our results suggested that curcumin down regulates MDR genes. Gene expression was decreased by 35.75, 31.30, 27.97 % for MDR1, LRP, BCRP respectively. In FLT3, it was 65.86 % for wild type and 31.79 % for FLT3-ITD. In addition to this, curcumin has also shown anti-proliferative effect as well as synergistic effect in combination with Cytarabine on primary leukemic cells. Thus, we can conclude that curcumin can be used as MDR modulator as well as chemosensitizer in combination with cytarabine, standard chemotherapeutic drug, to reduce the cytotoxicity profile as IC50 value decreases when treated in combination.

Evidence for circulating cancer stem-like cells and epithelial–mesenchymal transition phenotype in the pleurospheres derived from lung adenocarcinoma using liquid biopsy

Lung cancer stem cells are supposed to be the main drivers of tumor initiation, maintenance, drug resistance, and relapse of the disease. Hence, identification of the cellular and molecular aspects of these cells is a prerequisite for targeted therapy of lung cancer. Currently, analysis of circulating tumor cells has the potential to become the main diagnostic technique to monitor disease progression or therapeutic response as it is non-invasive. However, accurate detection of circulating tumor cells has remained a challenge, as epithelial cell markers used so far are not always trustworthy for detecting circulating tumor cells, especially during epithelial–mesenchymal transition. As cancer stem cells are the only culprit to initiate metastatic tumors, our aim was to isolate and characterize circulating tumor stem cells rather than circulating tumor cells from the peripheral blood of NSCLC adenocarcinoma as limited data are available addressing the gene expression profiling of lung cancer stem cells. Here, we reveal that CD44(+)/CD24(−) population in circulation not only exhibit stem cell–related genes but also possess epithelial–mesenchymal transition characteristics. In conclusion, the use of one or more cancer stem cell markers along with epithelial, mesenchymal and epithelial mesenchymal transition markers will prospectively provide the most precise assessment of the threat for recurrence and metastatic disease and has a great potential for forthcoming applications in harvesting circulating tumor stem cells and their downstream applications. Our results will aid in developing diagnostic and prognostic modalities and personalized treatment regimens like dendritic cell–based immunotherapy that can be utilized for targeting and eliminating circulating tumor stem cells, to significantly reduce the possibility of relapse and improve clinical outcomes.

Curcumin Targets Circulating Cancer Stem Cells by Inhibiting Self-Renewal Efficacy in Non-Small Cell Lung Carcinoma

BACKGROUND The ultimate goal of the study was to find a role of curcumin in targeting lung cancer stem cells by reducing their self-renewal efficiency causing DNA damage. MATERIALS AND METHODS Circulating lung cancer stem cells were isolated by sphere formation assay and further analysed by flow-cytometry and qRT-PCR for the presence of stem cell and stem cell transcription markers. The IC50 values of gemcitabine and curcumin were analysed by MTT assay, while curcumin induced DNA damage was scrutinized by single cell gel electrophoresis assay. RESULTS AND CONCLUSION Our results demonstrated that curcumin significantly affect the self-renewal ability of circulating lung cancer stem cells. The no. of spheres formed in the presence of curcumin was shown to be significantly decreased. Additionally, our results depicted that 4.52±0.72 % and 95.47±0.72 % (p < 0.0001) of DNA material was found to be present in head and tail, respectively, suggesting curcumins functional potential to cause DNA damage. Thus, we can conclude that curcumin can be used to target lung cancer stem cells which is responsible for the disease progression and metastasis by causing DNA damage or inhibiting their DNA repair mechanisms.

Circulating tumor stem like cells in oral squamous cell carcinoma: An unresolved paradox

OBJECTIVE Circulating tumor cells (CTCs) are increasingly gaining importance due to their immense potential in enhancing diagnosis, prognosis and response to therapy in solid malignancies. Therefore, we aimed to comprehend the molecular diversity and critical role of this disseminated tumor population in OSCC. METHODOLOGY CD44+ subpopulation was isolated using immuno-magnetic cell separation and their purity was validated using flow cytometry. Characterisation of self renewal potential and resistance to chemotherapy was assessed using tumor sphere forming and cytotoxicity assay. Gene expression profile of pertinent CSC (CD44s, CD44v3, CD44v6) and stemness markers (Bmi1 and Nanog) was carried out in CD44+ cells using Real Time PCR. Predominantly expressed markers and their association with clinico-pathological conditions were substantiated in 30 OSCC patients. RESULT Flow cytometry analysis depicted a predominant population of CD44+CD24-CD45- cells suggesting that circulating tumor cells had a subpopulation of CSC like cells in the circulation. These cells demonstrated increased sphere forming capability and intrinsic chemo-resistance compared to non-CSC, thus indicating the CSC features of self-renewal and chemo-resistance. Additionally, CD44+ cells showed significantly increased expression levels of CD44v6 and Nanog compared to CD44- cells. Clinically, expression pattern of CD44v6 and Nanog correlated with different anatomical subsites, loco-regional aggressiveness of the disease and recurrence, thus opening newer avenues that can be explored for better prognostic and therapeutic implications. CONCLUSION This study explored the inevitable role of CD44v6 and Nanog as circulating stem like cell markers in assessment of loco-regional aggressiveness, detection of relapse and therapeutic response and resistance.

Unravelling the link between embryogenesis and cancer metastasis

PURPOSE Cancer as opposed to embryonic development is characterized by dysregulated, uncontrolled and clonal growth of cells. Inspite of that they share certain commonality in gene expression patterns and a number of cellular & molecular features. Consequently, in the present study we aimed to evaluate the role of a definite set of genes in fetal liver, primary liver cancers and metastatic liver tissue. METHODS The relative expression of fourteen candidate genes obtained by data mining and manual curation of published data (CXCL12, CXCR4, CK7, CDH1, CTNNB1, CLDN4, VEGFA, HIF1A, MMP9, p53, OPN, CDKN2A, TGFBR2, MUC16, β-actin) were performed on 62 tissues (32 liver metastasis tissues and 30 primary Liver cancer tissues), Fetal liver tissues (below and above 20weeks of gestation) and 2 sets of control samples by real-time quantitative reverse transcription PCR (qRT-PCR). RESULTS Results showed significant down-regulation of MMP9 and TP53 in Fetal liver above 20weeks of gestation whereas it was up-regulated in fetal liver below 20weeks of gestation, primary liver cancers and liver metastasis. Contradictory to that OPN and CDKN2A were significantly up-regulated in primary liver cancer, liver metastasis; down-regulated in fetal liver above 20weeks of gestation but were not expressed during early embryo development (below 20weeks of gestation). Moreover, MMP9 and TP53 demonstrated a strong correlation with MUC16 whereas CDKN2A and OPN showed correlation with CXCL12/CXCR4 signifying that MUC16, CXCL12/CXCR4 might be involved in the complex process of cancer metastasis. CONCLUSION MMP9, OPN, TP53 and CDKN2A were the identified markers that were expressed in a similar pattern in early embryonic development and cancer development & invasion suggesting that these genes are activated during embryogenesis and might be re-expressed in cancer metastasis. Moreover, these genes govern a pathway that might be activated during cancer metastasis. Thus, targeting these molecules may provide better treatment for metastatic liver cancers.

Abstract 3060: Role of curcumin in targeting cancer-associated fibroblasts and modulation of tumor microenvironment in dendritic cell-based immunotherapy

INTRODUCTION: Cancer associated fibroblasts (CAFs), main component of tumor microenvironment (TME), modulate the recruitment and functions of tumor-associated immune cells by secreting various growth hormones, miRNAs and cytokines; thus having an important role in generation of immunosuppressive TME which is yet to be elucidated. Curcumin is known to have various properties including capability to modulate numerous target proteins including transcription factors, receptors, kinases, cytokines, enzymes and growth factors. Thus, aim of the study was to evaluate the effect of miRNAs and cytokines released by lung cancer patients’ derived CAFs and to assess immunomodulatory potential of curcumin on DC maturation by targeting these CAFs through modulating their TME. METHODOLOGY: CAFs were cultured from lung cancer patient derived tumor tissue biopsy and characterised using CAF-specific markers. Further, immature DCs (imDCs) were differentiated in presence of rGM-CSF and rIL-4 for 5-6 days. These imDCs were cultured in the presence of conditioned media derived from CAFs (CAFs-CM) as well as NFs (Normal Fibroblasts, NFs-CM) at day 6 for 48 hours to evaluate the effect of CAFs on DC maturation. Mature DCs (mDCs) were characterized by the presence of maturation markers CD80, CD83, CD86 and CTLA4 using qRT-PCR. Moreover, expression of miR-221, miR-222, miR-155, miR-142-3p and miR-146a was assessed to evaluate the role of epigenetic regulators on DC maturation. Cytokine profiling of CAFs-CM as well as CAFs-CM treated with curcumin was conducted. RESULTS: α-SMA+Vimentin+ cells were considered as CAFs. A significant upregulation of CD80, CD83 and CD86 was observed when cultured in the presence of NFs-CM while a remarkable downregulation of these markers was found when cultured in CAFs-CM. CTLA-4 was down regulated in NFs-CM as compared to CAFs-CM, suggesting the role of CAFs in generation of regulatory DCs. Amongst all miRNAs, miR-146a was shown to be up regulated dramatically in CAF-DCs (DCs cultured in CAFs-CM) as well as in CAFs-CM, suggesting the immunosuppressive role of miR-146a. Further, an increased expression of miR-146a was positively correlated with increased expression of anti-inflammatory cytokines like IL-6, IL-10, TGF-β and decreased expression of TNF-α (pro-inflammatory) in CM derived from CAF-DCs. Moreover, curcumin had the potential to convert regulatory DCs facilitated by CAFs into mDCs, which were characterized by high expression of co-stimulatory molecules, low expression of CTLA4, lower levels of immune suppressive cytokines production, lower levels of miR-146a. CONCLUSION: These findings provide insight into understanding the immunomodulatory role of curcumin in targeting CAFs and modulating the tumor microenvironment, thus enhancing antitumor immune response in DC based therapy. Citation Format: Sheefa Mirza, Nayan Jain, Rakesh Rawal. Role of curcumin in targeting cancer-associated fibroblasts and modulation of tumor microenvironment in dendritic cell-based immunotherapy [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 3060. doi:10.1158/1538-7445.AM2017-3060

Design and Synthesis of 1,4-dihydropyridine Derivatives as Anti-cancer Agent

AIMS A series of 1,4-dihydropyridine based compounds bearing benzylpyridinium moiety have been designed and evaluated for in vitro anticancer activity against glioblastoma U87MG, lung cancer A549 and colorectal adenocarcinoma Caco-2 cell lines using the MTT assay. METHOD Among these compounds, 7b, 7d, 7e, and 7f exhibited potent anticancer activity against the cell lines tested. The cytotoxicity of the synthesized derivatives was compared to standard drugs (carboplatin, gemcitabine, and daunorubicin). RESULT Thus, synthesized 1,4-dihydropyridines can be considered as the encouraging molecules for further drug development as anticancer agents.