Vaishali Kapoor

Erufosine simultaneously induces apoptosis and autophagy by modulating the Akt-mTOR signaling pathway in oral squamous cell carcinoma.

We investigated the anticancer activity of erufosine in oral squamous carcinoma cell lines in terms of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle and mTOR signaling pathway. Erufosine showed dose-dependent cytotoxicity in all cell lines, it induced autophagy as well as apoptosis, G2 cell cycle arrest and modulation of cyclin D1 expression. Further erufosine downregulated the phosphorylation of major components of mTOR pathway, like p-Akt at Ser473 and Thr308 residues, p-Raptor, p-mTOR, p-PRAS40 and its downstream substrates p-p70S6K and p-4EBP1 in a dose-dependent manner. The pre-treatment of tumor cells with p-mTOR siRNA increased cytotoxic effects of erufosine comparable to cisplatin but higher than rapamycin.

Antifungal and Antiproliferative Protein from Cicer arietinum: A Bioactive Compound against Emerging Pathogens

The emergence of epidemic fungal pathogenic resistance to current antifungal drugs has increased the interest in developing alternative antibiotics from natural sources. Cicer arietinum is well known for its medicinal properties. The aim of this work was to isolate antimicrobial proteins from Cicer arietinum. An antifungal protein, C-25, was isolated from Cicer arietinum and purified by gel filtration. C-25 protein was tested using agar diffusion method against human pathogenic fungi of ATCC strains and against clinical isolates of Candida krusei, Candida tropicalis, and Candida parapsilosis, and MIC values determined were varied from 1.56 to 12.5 μg/mL. The SEM study demonstrated that C-25 induces the bleb-like surface changes, irregular cell surface, and cell wall disruption of the fungi at different time intervals. Cytotoxic activity was studied on oral cancer cells and normal cells. It also inhibits the growth of fungal strains which are resistant to fluconazole. It reduced the cell proliferation of human oral carcinoma cells at the concentration of 37.5 μg/mL (IC50) and no toxic effect was found on normal human peripheral blood mononuclear cells even at higher concentration of 600 μg/mL. It can be concluded that C-25 can be considered as an effective antimycotic as well as antiproliferative agent against human oral cancer cells.

Deregulation of Beclin 1 in patients with tobacco-related oral squamous cell carcinoma

Autophagy is a physiologically regulated and evolutionary conserved process that plays a critical role in degradation of cytoplasmic proteins and other macromolecules within the lysosomes. Beclin-1, the mammalian orthologue of yeast Atg6, is an important mediator of autophagy that has been studied in many human cancers. However, the expression of Beclin-1 has not yet been investigated in oral cancer. We for the first time investigated the expression of Beclin-1 in serum and tissues and correlated it with the clinic-pathological features of oral cancer patients. m-RNA expression of Beclin-1 was evaluated in tumor and normal areas of surgical specimens from 10 oral cancer patients by real-time PCR. Approximately, 8-fold lower expression (p<0.001) of Beclin-1 mRNA was observed in tumor tissue as compared to the normal tissue. Serum levels of Beclin-1 were evaluated by SPR and ELISA. No significant difference was observed in serum Beclin-1 levels in patients as compared to healthy subjects, similarly no correlation was found between serum levels and clinic-pathological parameters such as stage, lymph node involvement and tumor size. Our results demonstrate that down-regulation of Beclin-1 may play an important role in the development and progression of oral cancer possibly by dysregulation of autophagy in tumor cells.

Structure Based Design and Synthesis of Peptide Inhibitor of Human LOX-12: In Vitro and In Vivo Analysis of a Novel Therapeutic Agent for Breast Cancer

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (KD) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39×10−8 M and 8.6×10−8 M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45×10−7 M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC50) of 75 µM and 400 µM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 µM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.

Genetic Polymorphisms in FAS (CD95) and FAS Ligand (CD178) Promoters and Risk of Tobacco-related Oral Carcinoma: Gene–Gene Interactions in High-risk Indians

We assessed the association of functional single nucleotide polymorphisms (SNP) in FAS -1377, -670 and FAS ligand (FASL) -844 promoters in 139 oral cancer patients and 126 normal subjects by PCR-RFLP. In logistic regression analysis FAS -1377 GA genotype appeared to marginally increase the risk while FASL -844 TC genotype appeared as low risk factor. The combined genotypes FAS -1377 GA or AA and FASL -844 TT (p <0.03), and FAS -670 AG or GG and FASL -844 TT (p <0.007) appeared to double the risk. FAS and FASL gene–gene and gene–environment interactions seems to modulate susceptibility/resistance to tobacco-related oral cancer in Indians.

Abnormal expression of PI3K isoforms in patients with tobacco-related oral squamous cell carcinoma

BACKGROUND The phosphatidylinositol 3-kinase (PI3K) signaling regulates several cellular functions such as motility, proliferation, angiogenesis and survival. METHODS Since there is no information on expression of PI3K isoforms in oral cancer, we studied the expression of different isoforms of PI3K (p110α, p110γ, PI3K-C2, Vps34p and p85α) in tumor samples and PBMC by RT and q-RTPCR and serum levels of PI3K p110α by SPR and ELISA techniques in 108 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 46 normal subjects. RESULTS We observed significantly higher PI3K p110α (p<0.0001) and lower (p<0.0001) vesicular sorting protein 34p (Vps34p) mRNA both in PBMC and tissue samples of oral cancer patients as compared to the normal controls. Other PI3K isoforms did not show such change. Circulating PI3K p110α levels were higher in patients (p<0.0001) as compared to healthy subjects, the SPR data showed direct correlation with advancing stage of the disease. PI3K p110α was overexpressed in tumor samples but not in the normal buccal mucosa. CONCLUSIONS Upregulation of circulating PI3K p110α isoform and its direct correlation with increasing tumor load in OSCC patients indicates that it may be a significant prognostic indicator and a suitable target for therapeutic/chemo-preventive strategies for tobacco-related OSCC.

Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers.

Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1. Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors. We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro. In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control.

Abstract 4599: Antibody targeting PDZ domain of TIP-1 induces proliferation arrest through AKT/mTOR signaling inhibition in lung cancer and glioblastoma

Antigens that are over-expressed in cancer in response to radiation are being used as novel targets. We showed tax interacting protein 1 (TIP-1) to be radiation-inducible that translocated to the surface of the cancer cell following irradiation. TIP-1, which consists of a single PDZ domain plays an important role in cell signaling, cancer development, and progression. TIP-1’s involvement in various survival pathways makes it an attractive target for anticancer therapeutics. We used antibodies specific to this PDZ domain to determine its role in cancer cell survival. We monitored proliferation in lung cancer (A549 and H460) and glioblastoma (D54 and U251) cells after 24, 48, 72 and 96h treatment with the anti-PDZ antibody. We observed a time-dependent proliferation arrest with anti-PDZ antibody treatment which was associated with increased apoptosis. The anti-PDZ antibody when combined with radiation (3Gy) led to reduced proliferation and colony formation. Anti-PDZ antibody had no effect on the proliferation of normal lung (MRC-5) and endothelial (HUVEC) cells. Cells treated with anti-PDZ antibody showed decreased levels of the phosphorylated forms of AKT, mTOR, and a downstream substrate of mTOR, 4EBP1. Anti-PDZ antibody treatment also led to an overall reduction in basal levels of AKT, mTOR, and 4EBP1. Further, we evaluated the effect of the anti-PDZ antibody on tumor growth in heterotopic mouse models of lung cancer (A549) and glioma (U251). We observed significant growth delay in mice treated with anti-PDZ antibody treatment when compared to mice treated with the isotype control. The combination of the anti-PDZ antibody with radiation showed an additive effect. Immunoblot analysis of tumor tissues also showed downregulation of phosphorylated and total levels of AKT, mTOR and 4EBP1 in the tumors treated with anti-PDZ antibody. Overall, our results suggest that TIP-1 is a promising therapeutic target for treatment of lung cancer and glioblastoma. Antibodies specific to the PDZ domain of TIP-1 enhance the efficacy of radiotherapy. The anti-PDZ antibodies need to be optimized further before translating it into the clinic. Citation Format: Vaishali Kapoor, David Dadey, Kelly Hoye, Andrea Collins, Dinesh Thotala, Dennis Hallahan. Antibody targeting PDZ domain of TIP-1 induces proliferation arrest through AKT/mTOR signaling inhibition in lung cancer and glioblastoma [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 4599. doi:10.1158/1538-7445.AM2017-4599

Erufosine Induces Autophagy and Apoptosis in Oral Squamous Cell Carcinoma: Role of the Akt–mTOR Signaling Pathway

The novel alkylphosphocholine erufosine exerts selective antitumor activity, but no myelotoxicity. Its mechanism of action is not fully understood, but is related to dephosphorylation of Akt and Rb. Our study revealed a novel mechanism underlying the anticancer activity of erufosine via downregulation of the mammalian target of rapamycin (mTOR) signaling cascade in oral squamous cell carcinoma cell lines. Erufosine showed dose-dependent cytotoxicity and simultaneously induced both autophagy and apoptosis via conversion of microtubule-associated protein light chain 3B-I (LC3B-I) to LC3B-II, caspase-3/7 activation, and Poly(ADP-ribose) polymerase (PARP) cleavage, respectively, in all the cell lines. In addition, G2 cell cycle arrest was observed along with cyclin D1 downregulation. These effects of erufosine were due to modulation of the mTOR signaling pathway. It readily downregulated p-Akt, p-Raptor, p-mTOR, and its downstream substrates, p-p70S6K and p-4EBP1. p-PRAS40 was reduced to undetectable levels. Moreover, blockage of p-mTOR expression by small interference RNA (siRNA) increased tumor cell sensitivity to erufosine, which was comparable to cisplatin, but higher than that of rapamycin. Furthermore, it showed additive effects on tumor cell cytotoxicity in combination with 5-flourouracil and cisplatin. Our study therefore indicated that erufosine induces apoptosis, autophagy, cell cycle arrest, and downregulates mTOR signaling in oral cancer cell lines. It has potential as an anticancer drug either alone or in combination with other chemotherapeutic agents.

Abstract 913: Erufosine simultaneously induces apoptosis and autophagy by modulating the mTOR signaling pathway in oral squamous cell carcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Erufosine (erucylphospho-N, N, N-trimethylpropylammonium) is an i.v. injectable alkylphosphocholine analog, which is active against various hematological malignancies in vitro. In this study, we reveal activity in oral squamous carcinoma cell lines and down-regulation of the mTOR signaling cascade as novel mechanism underlying the anticancer activity of erufosine. To that purpose, four oral squamous cell carcinoma cell lines (Cal27, FaDu, SCC9 and SCC25) were exposed to erufosine and assessed for modulation of cell proliferation, colony formation, induction of autophagy/apoptosis, cell cycle distribution, and signaling of the mTOR pathway. In addition, combination effects of erufosine with decreased p-mTOR expression or cytotoxic drugs were investigated. Erufosine showed dose-dependent cytotoxicity in all cell lines as observed by MTT and clonogenicity assays. It simultaneously induced both autophagy and apoptosis via conversion of LC3BI to LC3BII, caspase 3/7 activation and PARP cleavage, respectively. In addition, G2 cell cycle arrest was observed along with cyclin D1 down-regulation. Subsequently, we observed that erufosine readily down-regulated in a dose-dependent manner the phosphorylation of components of the mTOR signaling pathway, like p-Akt, p-Raptor, p-mTOR, and its downstream substrates p-p70S6K and p-4EBP1. p-PRAS40 was reduced to undetectable levels at the highest concentrations of erufosine. Moreover, blockage of p-mTOR expression by siRNA, increased tumor cell sensitivity to erufosine and the resulting antiproliferative effect was comparable to the respective combination with cisplatin but superior to that with rapamycin. Further, erufosine showed additive tumor cell cytotoxicity in combination with 5-flourouracil and cisplatin. In conclusion, these data indicate that erufosine induces apoptosis, autophagy, cell cycle arrest and down-regulates mTOR signaling in oral cancer cell lines. Thus, it has potential as an anti-oral cancer drug either alone or in combination with other chemotherapeutic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 913. doi:1538-7445.AM2012-913