Aditya Parekh

Molecular targeting of Akt by thymoquinone promotes G1 arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells

AIM Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQs mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells. MAIN METHODS MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies. KEY FINDINGS Studies revealed G1 phase arrest till 24h incubation with TQ while extended exposure showed phase shift to subG1 indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K. SIGNIFICANCE Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.

Celecoxib alleviates tamoxifen-instigated angiogenic effects by ROS-dependent VEGF/VEGFR2 autocrine signaling

BackgroundTamoxifen (TAM) is widely used in the chemotherapy of breast cancer and as a preventive agent against recurrence after surgery. However, extended TAM administration for breast cancer induces increased VEGF levels in patients, promoting new blood vessel formation and thereby limiting its efficacy. Celecoxib (CXB), a selective COX-2 inhibitor, suppresses VEGF gene expression by targeting the VEGF promoter responsible for its inhibitory effect. For this study, we had selected CXB as non-steroidal anti-inflammatory drug in combination with TAM for suppressing VEGF expression and simultaneously reducing doses of both the drugs.MethodsThe effects of CXB combined with TAM were examined in two human breast cancer cell lines in culture, MCF7 and MDA-MB-231. Assays of proliferation, apoptosis, angiogenesis, metastasis, cell cycle distribution, and receptor signaling were performed.ResultsHere, we elucidated how the combination of TAM and CXB at nontoxic doses exerts anti-angiogenic effects by specifically targeting VEGF/VEGFR2 autocrine signaling through ROS generation. At the molecular level, TAM-CXB suppresses VHL-mediated HIF-1α activation, responsible for expression of COX-2, MMP-2 and VEGF. Besides low VEGF levels, TAM-CXB also suppresses VEGFR2 expression, confirmed through quantifying secreted VEGF levels, luciferase and RT-PCR studies. Interestingly, we observed that TAM-CXB was effective in blocking VEGFR2 promoter induced expression and further 2 fold decrease in VEGF levels was observed in combination than TAM alone in both cell lines. Secondly, TAM-CXB regulated VEGFR2 inhibits Src expression, responsible for tumor progression and metastasis. FACS and in vivo enzymatic studies showed significant increase in the reactive oxygen species upon TAM-CXB treatment.ConclusionsTaken together, our experimental results indicate that this additive combination shows promising outcome in anti-metastatic and apoptotic studies. In a line, our preclinical studies evidenced that this additive combination of TAM and CXB is a potential drug candidate for treatment of breast tumors expressing high levels of VEGF and VEGFR2. This ingenious combination might be a better tailored clinical regimen than TAM alone for breast cancer treatment.

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.

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.

Glucose Directly Promotes Antifungal Resistance in the Fungal Pathogen, Candida spp.

Background: Glucose level alters susceptibility of antifungal agents during chemotherapy in diabetes patients. Results: Glucose selectively interacts with antifungal agents, strongly affects azole drugs, and forms complexes by hydrogen bonding. Conclusion: It is important for researchers and pharmaceuticals to make new antibiograms for diabetes patients. Significance: Drug selection is important for controlling fungal infections in diabetes patients. Effects of glucose on the susceptibility of antifungal agents were investigated against Candida spp. Increasing the concentration of glucose decreased the activity of antifungal agents; voriconazole was the most affected drugs followed by amphotericin B. No significant change has been observed for anidulafungin. Biophysical interactions between antifungal agents with glucose molecules were investigated using isothermal titration calorimetry, Fourier transform infrared, and 1H NMR. Glucose has a higher affinity to bind with voriconazole by hydrogen bonding and decrease the susceptibility of antifungal agents during chemotherapy. In addition to confirming the results observed in vitro, theoretical docking studies demonstrated that voriconazole presented three important hydrogen bonds and amphotericin B presented two hydrogen bonds that stabilized the glucose. In vivo results also suggest that the physiologically relevant higher glucose level in the bloodstream of diabetes mellitus mice might interact with the available selective agents during antifungal therapy, thus decreasing glucose activity by complex formation. Thus, proper selection of drugs for diabetes mellitus patients is important to control infectious diseases.

Therapeutic implication of ‘Iturin A’ for targeting MD-2/TLR4 complex to overcome angiogenesis and invasion

Tumor angiogenesis and invasion are deregulated biological processes that drive multistage transformation of tumors from a benign to a life-threatening malignant state activating multiple signaling pathways including MD-2/TLR4/NF-κB. Development of potential inhibitors of this signaling is emerging area for discovery of novel cancer therapeutics. In the current investigation, we identified Iturin A (A lipopeptide molecule from Bacillus megaterium) as a potent inhibitor of angiogenesis and cancer invasion by various in vitro and in vivo methods. Iturin A was found to suppress VEGF, a powerful inducer of angiogenesis and key player in tumor invasion, as confirmed by ELISA, western blot and real time PCR. Iturin A inhibited endothelial tube arrangement, blood capillary formation, endothelial sprouting and vascular growth inside the matrigel. In addition, Iturin A inhibited MMP-2/9 expression in MDA-MB-231 and HUVEC cells. Cancer invasion, migration and colony forming ability were significantly hampered by Iturin A. Expressions of MD-2/TLR4 and its downstream MyD88, IKK-α and NF-κB were also reduced in treated MDA-MB-231 and HUVEC cells. Western blot and immunofluorescence study showed that nuclear accumulation of NF-κB was hampered by Iturin A. MD-2 siRNA or plasmid further confirmed the efficacy of Iturin A by suppressing MD-2/TLR4 signaling pathway. The in silico docking study showed that the Iturin A interacted well with the MD-2 in MD-2/TLR4 receptor complex. Conclusively, inhibition of MD-2/TLR4 complex with Iturin A offered strategic advancement in cancer therapy.

Wavelet-based multiscale analysis of bioimpedance data measured by electric cell-substrate impedance sensing for classification of cancerous and normal cells.

The paper presents a study to differentiate normal and cancerous cells using label-free bioimpedance signal measured by electric cell-substrate impedance sensing. The real-time-measured bioimpedance data of human breast cancer cells and human epithelial normal cells employs fluctuations of impedance value due to cellular micromotions resulting from dynamic structural rearrangement of membrane protrusions under nonagitated condition. Here, a wavelet-based multiscale quantitative analysis technique has been applied to analyze the fluctuations in bioimpedance. The study demonstrates a method to classify cancerous and normal cells from the signature of their impedance fluctuations. The fluctuations associated with cellular micromotion are quantified in terms of cellular energy, cellular power dissipation, and cellular moments. The cellular energy and power dissipation are found higher for cancerous cells associated with higher micromotions in cancer cells. The initial study suggests that proposed wavelet-based quantitative technique promises to be an effective method to analyze real-time bioimpedance signal for distinguishing cancer and normal cells.

Bioimpedimetric analysis in conjunction with growth dynamics to differentiate aggressiveness of cancer cells

Determination of cancer aggressiveness is mainly assessed in tissues by looking at the grade of cancer. There is a lack of specific method to determine aggressiveness of cancer cells in vitro. In our present work, we have proposed a bio-impedance based non-invasive method to differentiate aggressive property of two breast cancer cell lines. Real-time impedance analysis of MCF-7 (less aggressive) and MDA-MB-231 cells (more aggressive) demonstrated unique growth pattern. Detailed slope-analysis of impedance curves at different growth phases showed that MDA-MB-231 had higher proliferation rate and intrinsic resistance to cell death, when allowed to grow in nutrient and space limiting conditions. This intrinsic nature of death resistance of MDA-MB-231 was due to modulation and elongation of filopodia, which was also observed during scanning electron microscopy. Results were also similar when validated by cell cycle analysis. Additionally, wavelet based analysis was used to demonstrate that MCF-7 had lesser micromotion based cellular activity, when compared with MDA-MB-231. Combined together, we hypothesize that analysis of growth rate, death resistance and cellular energy, through bioimpedance based analysis can be used to determine and compare aggressiveness of multiple cancer cell lines. This further opens avenues for extrapolation of present work to human tumor tissue samples.

Multi-nucleated cells use ROS to induce breast cancer chemo-resistance in vitro and in vivo

Although there is a strong correlation between multinucleated cells (MNCs) and cancer chemo-resistance in variety of cancers, our understanding of how multinucleated cells modulate the tumor micro-environment is limited. We captured multinucleated cells from triple-negative chemo-resistant breast cancers cells in a time frame, where they do not proliferate but rather significantly regulate their micro-environment. We show that oxidatively stressed MNCs induce chemo-resistance in vitro and in vivo by secreting VEGF and MIF. These factors act through the RAS/MAPK pathway to induce chemo-resistance by upregulating anti-apoptotic proteins. In MNCs, elevated reactive oxygen species (ROS) stabilizes HIF-1α contributing to increase production of VEGF and MIF. Together the data indicate, that the ROS-HIF-1α signaling axis is very crucial in regulation of chemo-resistance by MNCs. Targeting ROS-HIF-1α in future may help to abrogate drug resistance in breast cancer.

201 Combinatorial Effect of ZD6474 and Thymoquinone Inhibits Src Mediated ERK-1/2/STAT3 Signalling and Renders Antimetastasis in Breast Cancer

Results: NZ-8 was generated, and reacted with both human glioblastoma cell line LN319 and human mesothelioma cell line H226 in flow cytometry. KD of NZ-8 was determined to be 7.6×10−8 M, whereas KD of NZ-1 was determined to be 7.2×10−9 M. Both ADCC and CDC were induced by NZ8 against LN319 cells. NZ-8 inhibited platelet aggregation by CHO/hPDPN in a dose-dependent manner. In addition, podoplanin-induced pulmonary metastasis was suppressed by NZ-8 dose-dependently. Antitumor activity of NZ-8 was evaluated by treating nude mice with NZ-8 after subcutaneous injection of CHO/hPDPN. The subcutaneous tumor formation rate was 10/10 (100%) for the control group and 4/10 (40%) for the NZ-8 antibodyadministered group. Subcutaneous tumors were significantly smaller in the NZ-8 antibody-administered group, indicating that administration of NZ-8 significantly inhibited the growth of CHO/hPDPN cells in vivo. Conclusions: NZ-8 possesses the potent and therapeutic antitumor effects based on ADCC and CDC against podoplanin-expressing cells. Targeting podoplanin by using the NZ-8 antibody might be useful as a novel immunotherapy against podoplanin-expression tumors.