Sheetal Parida

Abstract 3685: Managing cervical cancer using multifunctional gold nanorods

Introduction: Treatment of, cervical cancer, the third most prevalent cancer in women, is challenged by resistance to chemo and radio therapy, organ toxicities and disease relapse which is mainly due to undiagnosed small metastases and failure in monitoring the course of treatment. Imaging is therefore another critical aspect in overall cancer management. We designed a triple combination treatment modality involving photo thermal therapy, EGFR mAb and GW627368X, an EP4 prostanoid receptor antagonist in a single nanoprobe which also serves as an efficient X-ray CT contrast agents. Methodology: Stimuli responsive, GW627368X loaded, EGFR mAb tagged gold nanorods(GNRs) were prepared by seed mediated method and characterized by TEM, NMR, IR and UV absorption spectroscopy. Cellular uptake of nanoparticles was studied by flow cytometry and fluorescent imaging. Drug release kinetics was studied prior to in vitro study. Cervical cancer cells, SiHa and ME180, were incubated with GNRs for 45mins, irradiated with cw-NIR laser (808nm) for various time periods and incubated for 12 hours. Cellular effects were studied by viability assays, flow cytometry, florescent staining, SEM, ELISA and western blot analysis. The X-ray attenuation property of nanoprobe was demonstrated by CT imaging of GNR incubated cervical cancer cells compared with untreated cells. Results: Due to surface plasmone resonance of GNRs, energy from incident NIR rays are converted into heat inducing apoptosis evident in viability assays further confirmed by flow cytometry, microscopy and western blot. Accumulation of the GNRs around the EGFR overexpressing transformed cells and subsequent uptake was confirmed microscopically and by increase in SSC of cells. EGFR blockade disrupts ligand receptor interaction down regulating survival pathways like Ras/MAPK, PI3K/Akt and JAK/STAT. Stimuli dependent drug release inhibits EP4 receptor in turn down regulating COX-2, PI3K/Akt and angiogenesis. Reduced VEGF, EGF and PGE-2 production was confirmed by ELISA and down regulation of pAkt, pMAPK, pEGFR, COX-2 and EP4 proteins was confirmed by western blot analysis. Prominently distinguishable CT attenuation was obtained with gold nanoprobes compared to untreated cells. Conclusion: EGFR mAb tagged, GW627368X loaded GNRs is novel therapeutic approach merging multiple strategies in single nanoprobe. Besides targeted accumulation, EGFR mAb disrupts EGFR signaling which along with stimuli dependent drug release block multiple downstream pathways. Photothermal therapy is highly efficient, selective approach as NIR has high tissue permeability but is harmless to normal tissue. Moreover, high X-ray atteneuation of gold makes it suitable a CT contrast agent for both diagnosis and monitoring the course of treatment. The proposed modality would thus prove to be highly efficient in overall management of cervical cancer. Citation Format: Sheetal Parida, Mahitosh Mandal. Managing cervical cancer using multifunctional gold nanorods. [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 3685. doi:10.1158/1538-7445.AM2015-3685

Polymer grafted magnetic nanoparticles for delivery of anticancer drug at lower pH and elevated temperature

Efficient and controlled delivery of therapeutics to tumor cells is one of the important issues in cancer therapy. In the present work, a series of pH- and temperature-responsive polymer grafted iron oxide nanoparticles were prepared by simple coupling of aminated iron oxide nanoparticle with poly(N-isopropylacrylamide-ran-poly(ethylene glycol) methyl ether acrylate)-block-poly(acrylic acid) (P(NIPA-r-PEGMEA)-b-PAA). For this, three water soluble block polymers were prepared via reversible addition fragmentation transfer (RAFT) polymerization technique. At first, three different block copolymers were prepared by polymerizing mixture of NIPA and PEGMEA (with varying mole ratio) in presence of poly(tert-butyl acrylate) (PtBA) macro chain transfer agent. Subsequently, P(NIPA-r-PEGMEA)-b-PAA copolymers were synthesized by hydrolyzing tert-butyl acrylate groups of the P(NIPA-r-PEGMEA)-b-PtBA copolymers. The resulting polymers were then grafted to iron oxide nanoparticles, and these functionalized nanoparticles were thoroughly characterized by X-ray diffraction (XRD), thermogravimetric analysis (TGA), zeta potential measurements, transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), vibrating sample magnetometer (VSM) and Fourier transform infrared spectroscopy (FTIR). Doxorubicin (DOX), an anti-cancer drug, was loaded into the polymer coated nanoparticles and its release behavior was subsequently studied at different pH and temperatures. The drug release pattern revealed a sustained release of DOX preferentially at the desired lysosomal pH of cancer cells (pH 5.0) and slightly above the physiological temperature depending upon the composition of the copolymers. The potential anticancer activity of the polymer grafted DOX loaded nanoparticles were established by MTT assay and apoptosis study of cervical cancer ME 180cells in presence of the nanoparticles. Thus, these particles can be utilized for controlled delivery of anticancer drugs at the desired lysosomal pH and/or by slightly heating the cells using magnetic hyperthermia.

Gold nanorod embedded reduction responsive block copolymer micelle-triggered drug delivery combined with photothermal ablation for targeted cancer therapy.

BACKGROUND Gold nanorods, by virtue of surface plasmon resonance, convert incident light energy (NIR) into heat energy which induces hyperthermia. We designed unique, multifunctional, gold nanorod embedded block copolymer micelle loaded with GW627368X for targeted drug delivery and photothermal therapy. METHODS Glutathione responsive diblock co-polymer was synthesized by RAFT process forming self-assembled micelle on gold nanorods prepared by seed mediated method and GW627368X was loaded on to the reduction responsive gold nanorod embedded micelle. Photothermal therapy was administered using cwNIR laser (808nm; 4W/cm2). Efficacy of nanoformulated GW627368X, photothermal therapy and combination of both were evaluated in vitro and in vivo. RESULTS In response to photothermal treatment, cells undergo regulated, patterned cell death by necroptosis. Combining GW627368X with photothermal treatment using single nanoparticle enhanced therapeutic outcome. In addition, these nanoparticles are effective X-ray CT contrast agents, thus, can help in monitoring treatment. CONCLUSION Reduction responsive nanorod embedded micelle containing folic acid and lipoic acid when treated on cervical cancer cells or tumour bearing mice, aggregate in and around cancer cells. Due to high glutathione concentration, micelles degrade releasing drug which binds surface receptors inducing apoptosis. When incident with 808nm cwNIR lasers, gold nanorods bring about photothermal effect leading to hyperthermic cell death by necroptosis. Combination of the two modalities enhances therapeutic efficacy by inducing both forms of cell death. GENERAL SIGNIFICANCE Our proposed treatment strategy achieves photothermal therapy and targeted drug delivery simultaneously. It can prove useful in overcoming general toxicities associated with chemotherapeutics and intrinsic/acquired resistance to chemo and radiotherapy.

Thymoquinone restores radiation-induced TGF-β expression and abrogates EMT in chemoradiotherapy of breast cancer cells

Radiotherapy remains a prime approach to adjuvant therapies in patients with early and advanced breast cancer. In spite of therapeutic success, metastatic progression in patients undergoing therapy, limits its application. However, effective therapeutic strategies to understand the cellular and molecular machinery in inhibiting radiation‐induced metastatic progression, which is poorly understood so far, need to be strengthened. Ionizing radiation was known to prompt cancer cells metastatic ability by eliciting Transforming Growth Factor‐beta (TGF‐β), a key regulator in epithelial–mesenchymal transdifferentiation and radio‐resistance. In this viewpoint, we employed thymoquinone as a radiosensitizer to investigate its migration and invasion reversal abilities in irradiated breast cancer cell lines by assessing their respective attributes. The role of metastasis regulatory molecules like TGF‐β, E‐cadherin, and integrin αV and its downstream molecules were determined using RT‐PCR, western blotting, immunofluorescence, and extracellular TGF‐β levels affirmed through ELISA assays. These studies affirmed the TGF‐β restoring ability of thymoquinone in radiation‐driven migration and invasion. Also, results demonstrated that the epithelial markers E‐cadherin and cytokeratin 19 were downregulated whereas mesenchymal markers like integrin αV, MMP9, and MMP2 were upregulated by irradiation treatment; however thymoquinone pre‐sensitization has reverted the expression of these proteins back to control proteins expression. Here, paclitaxel was chosen as an apoptosis inducer in TGF‐β restored cells and confirmed its cytotoxic effects in radiation alone and thymoquinone sensitized irradiated cells. We conclude that this therapeutic modality is effective in preventing radiation‐induced epithelial–mesenchymal transdifferentiation and concomitant induction of apoptosis in breast cancer. J. Cell. Physiol. 230: 620–629, 2015.

Inflammation induced by human papillomavirus in cervical cancer and its implication in prevention.

Many pathological conditions including most cancers show an exacerbated activation of the inflammatory pathways and their sustained maintenance. In cervical carcinogenesis, the hyperactivation of the inflammatory pathways plays an important role in tumorigenesis, progression of the disease from low-grade lesions to invasive cervical cancer as well as in the initiation of other infections such as HIV. Cyclooxygenase-2 (COX-2) is the inducible isoform of cyclooxygenases regulated by growth factors and cytokines, hence overexpressed under inflammatory conditions. Higher levels of COX-2 expression are closely related to a higher incidence of parametrial invasion and lymph node metastases in early-stage uterine cervical cancer. The principal products of COX-2 enzyme, prostanoids, are released from cells and act locally in autocrine and paracrine modes, activating diverse intracellular pathways, which in turn induce cellular proliferation, antiapoptotic activity, angiogenesis, and increased metastasis. In the current review, we focus on the role of the viral oncogenic proteins in activation of the COX-2/PGE2 pathway and their clinical implications, a better understanding of which would be helpful in designing newer and more effective therapeutic and preventive strategies for the disease.

pH-degradable and thermoresponsive water-soluble core cross-linked polymeric nanoparticles as potential drug delivery vehicle for doxorubicin

Controlled and efficient delivery of therapeutics to tumor cells is one of the key issues in cancer therapy. In the present work, a new class of water soluble core cross-linked polymer nanoparticles (CLPNs) possessing acid degradable core and thermoresponsive shell was synthesized for pH-triggered delivery of drugs to cancerous cells. The diol groups of the poly(ethylene glycol)-b-poly(N-isopropylacrylamide)-b-poly(glycidyl methacrylate) diol triblock copolymer were utilized to form the core cross-linked polymeric nanoparticles through an arm-first method by reaction with aldehyde functionalized cross-linkers through formation of acetal linkages. The encapsulation efficiency as well as the release properties of these CLPNs was investigated using doxorubicin (DOX), a known anticancer drug. The release was found to be preferable at the desired lysosomal pH (∼5.0) of the cancer cells and below the LCST (∼32 °C) of poly(N-isopropylacrylamide) (PNIPA). The cytotoxicities of the precursor polymer as well as the CLPNs were tested on the growth of NIH/3T3, normal mouse fibroblast cells, and they were found to be nontoxic. The anticancer activity of the DOX loaded CLPN was confirmed using cervical cancer cell lines HeLa and SiHa by MTT assay, morphological studies and flow cytometry. These studies revealed an increased accumulation of the drug around the nucleus when treated with DOX-loaded CLPN as compared to free DOX along with significant reduction in IC50 of both the cell lines. Thus, these CLPNs are potentially useful for controlled drug delivery in the case of advanced chemotherapeutic applications.

Molecular inhibition of prostaglandin E2 with GW627368X: Therapeutic potential and preclinical safety assessment in mouse sarcoma model

Prostaglandin E2, the major COX-2 product, acts via 4 functionally distinct prostanoid receptors, EP(1–4). PGE-2, through its receptors, feeds back to positively increase COX-2 expression augmenting its own synthesis thereby driving angiogenesis, while suppressing apoptosis and innate immunity. In addition to the well characterized PGE2/EP4/cAMP/PKA/CREB, EP4 activation increases GSK3 phosphorylation via PI3K and Akt consequently reducing β-catenin phosphorylation. EP4 induces angiogenesis by enhancing VEGF production via ERK activation. These effects of EP4 are asserted either directly or via EGFR transactivation depending on the type of cancer. In view of the safety concerns regarding long term use of COX-2 inhibitors and to find more effective alternatives, we evaluated the potential of EP4 prostanoid receptor as a target for treating cancer progression using a highly selective EP4 antagonist, 4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide. Oral administration of GW627368X showed significant tumor regression characterized by tumor reduction and induction of apoptosis. Reduction in prostaglandin E2 synthesis also led to reduced level of VEGF in plasma. Regulation of multiple pathways downstream of EP4 was evident by down regulation of COX-2, p-Akt, p-MAPK and p-EGFR. Considering wide distribution of the EP4 prostanoid receptor in major organs and the array of physiological processes it contributes to, the safety profile of the drug was analyzed. No major organ toxicity, immunosupression, behavioral change or change in blood parameters attributable to the drug was observed. The results assert the significance of EP4 prostanoid receptor as a therapeutic target as well as the safety of EP4 blockade by GW627368X.

Blockade of autophagy enhances proapoptotic potential of BI-69A11, a novel Akt inhibitor, in colon carcinoma

BI-69A11, novel Akt inhibitor, is currently drawing much attention due to its intriguing effect in inducing apoptosis in melanoma, breast, prostate and colon cancer. However, earlier reports reveal that PI3K/Akt/mTOR inhibitors promote autophagy at the early stage as a survival mechanism that might affect its apoptotic potential. It is necessary to investigate whether BI-69A11 mediated apoptosis is associated with autophagy for enhancing its therapeutic efficacy. Here, we found that BI-69A11 induced autophagy at earlier time point through the inhibition of Akt/mTOR/p70S6kinase pathway. Dose-dependent and time-dependent conversion of LC3-I to LC3-II, increased accumulation of LC3-GFP dots in cytoplasm and increase in other autophagic markers such as Beclin-1, firmly supported the fact that BI-69A11 induces autophagy. Atg5, Atg7 and Beclin-1 siRNA mediated genetic attenuation and pre-treatment with pharmacological inhibitor 3-MA and CQ diminished the autophagy and increased the propensity of cell death towards apoptosis. It was also suggested that BI-69A11 mediated interaction between Akt, HSP-90 and Beclin-1 maintained the fine balance between autophagy and apoptosis. Interaction between Beclin-1 and HSP90 is one of the prime causes of induction of autophagy. Here, we also generated a novel combination therapy by pretreatment with CQ that inhibited the autophagy and accelerated the apoptotic potential of BI-69A11. In summary; our findings suggest that induction of autophagy lead to the resistance of colon cancer towards BI-69A11 mediated apoptosis.

BI2536 - A PLK inhibitor augments paclitaxel efficacy in suppressing tamoxifen induced senescence and resistance in breast cancer cells

Tamoxifen resistance is a multifaceted phenomenon, characterized by the constitutive activation of multiple signaling cascades that provide an additional survival advantage to cells. Ground studies related to reverse the tamoxifen resistance by employing chemotherapeutic drugs that specifically inhibit proteins, those of aberrantly expressed, are required. Seminal studies showed that p38 signaling and VEGF play crucial role in acquiring resistance to tamoxifen. In this view, we had chosen paclitaxel, a mitotic inhibitor with anti-proliferative effects against a wide array of cancers in this study. Further to mitigate the undesirable complications of paclitaxel (PAC), we employed this drug in combination along with BI2536 (BI), a PLK inhibitor for this study to sensitize the tamoxifen resistant cells to apoptosis. MCF 7/TAM and T-47D/TAM cells were treated with PAC, BI and in combination (BI-PAC) evaluated for its anticancer activity through apoptotic and western blot analysis. Modulatory effects of BI-PAC on p38 inactivation were affirmed through immunofluorescence and drug potential studies. Results reveal that cells were subjected to apoptosis on drug(s) treatment which was confirmed through cytotoxicity, annexin studies. Further, the anti-proliferative effects of the drug(s) were affirmed through nuclear morphological and TUNEL assays. Immunoblot results revealed the upregulation of proapoptotic Bax, cleaved caspase 9 along with Bcl-2, MDM2, Cox-2, and P-Gly down regulation after 24h drug treatments. Moreover, phospho studies further construed the rationale behind the apoptosis and deduced the inactivation of p38 and NF-κB role in inducing apoptosis in drug treated cells. The efficacy of drug combinations in inactivating p38 was evaluated through drug potential studies. Further, BI-PAC treatments showed inhibition of p38 mediated senescence in tamoxifen resistant cells. Overall, our observations provide a new therapeutic combination that sensitizes tamoxifen resistant cells to apoptosis by specifically targeting p38 signaling and its downstream molecules and subsequently reduces extracellular VEGF levels.

Cooperative effect of BI-69A11 and celecoxib enhances radiosensitization by modulating DNA damage repair in colon carcinoma

Amplification of PI3K-Akt pathway promotes radioresistance in various cancers including colorectal carcinoma. Local recurrence in colon cancer causes poor prognosis affecting overall survival of cancer-affected patient population. To avoid local recurrence, pre-operative or post-operative additional radiotherapy is given. However, main concern regarding radiotherapy is to increase the radiosensitivity of malignant cell without hampering the activities of normal cells. In this context, addition of two or more than two chemotherapeutic drugs as a radiosensitizer is a common practice in radiation biology. BI-69A11 earlier showed potential apoptosis-inducing effect in melanoma and colon carcinoma. Celecoxib showed anti-cancer effects in both COX-2 dependent and independent pathways and used to act as a radiosensitizing enhancer. Here, we suggest that the combination of BI-69A11 and celecoxib inhibits the phosphorylation of ataxia telangiectasia mutated (ATM) kinase and DNA-PK responsible for ionizing radiation (IR)-induced double-strand break (DSB) repair. Moreover, the combinatorial effect of BI-69A11 and celecoxib attenuates the IR-induced G2/M cell cycle arrest. Furthermore, this combination also impairs IR-induced activation of Akt and downstream targets of ATM. This might lead to induced activation of apoptotic pathway after triple therapy treatment modulating pro-apoptotic and anti-apoptotic proteins. This activation of apoptotic pathway also showed the interdependence of PUMA and BAD in triple combination-treated colon cancer cells in a p53 independent manner. This study reveals the therapeutic potential of the triple combination therapy in prevention of radioresistance. Besides, it also demonstrates the cytotoxic effects of triple combination therapy in colon cancer. This study shows utility and potential implication on safety of the patients undergoing radiation therapy.