Browsing by Author "Kalainayakan, Sarada Preeta"
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Item Broadband Transient Absorption Study of Photoexcitations in Lead Halide Perovskites: Towards a Multiband Picture(2016-02-24) Alam, Md Maksudul; Sohoni, Sagar; Kalainayakan, Sarada Preeta; Garrossian, Massoud; Zhang, Li; 0000-0001-9242-0763 (Zhang, L); Alam, Md Maksudul; Sohoni, Sagar; Kalainayakan, Sarada Preeta; Zhang, LiBACKGROUND: Aberrant Hedgehog (Hh) signaling is associated with the development of many cancers including prostate cancer, gastrointestinal cancer, lung cancer, pancreatic cancer, ovarian cancer, and basal cell carcinoma. The Hh signaling pathway has been one of the most intensely investigated targets for cancer therapy, and a number of compounds inhibiting Hh signaling are being tested clinically for treating many cancers. Lung cancer causes more deaths than the next three most common cancers (colon, breast, and prostate) combined. Cyclopamine was the first compound found to inhibit Hh signaling and has been invaluable for understanding the function of Hh signaling in development and cancer. To find novel strategies for combating lung cancer, we decided to characterize the effect of cyclopamine tartrate (CycT), an improved analogue of cyclopamine, on lung cancer cells and its mechanism of action. METHODS: The effect of CycT on oxygen consumption and proliferation of non-small-cell lung cancer (NSCLC) cell lines was quantified by using an Oxygraph system and live cell counting, respectively. Apoptosis was detected by using Annexin V and Propidium Iodide staining. CycT’s impact on ROS generation, mitochondrial membrane potential, and mitochondrial morphology in NSCLC cells was monitored by using fluorometry and fluorescent microscopy. Western blotting and fluorescent microscopy were used to detect the levels and localization of Hh signaling targets, mitochondrial fission protein Drp1, and heme-related proteins in various NSCLC cells. RESULTS: Our findings identified a novel function of CycT, as well as another Hh inhibitor SANT1, to disrupt mitochondrial function and aerobic respiration. Our results showed that CycT, like glutamine depletion, caused a substantial decrease in oxygen consumption in a number of NSCLC cell lines, suppressed NSCLC cell proliferation, and induced apoptosis. Further, we found that CycT increased ROS generation, mitochondrial membrane hyperpolarization, and mitochondrial fragmentation, thereby disrupting mitochondrial function in NSCLC cells. CONCLUSIONS: Together, our work demonstrates that CycT, and likely other Hh signaling inhibitors, can interrupt NSCLC cell function by promoting mitochondrial fission and fragmentation, mitochondrial membrane hyperpolarization, and ROS generation, thereby diminishing mitochondrial respiration, suppressing cell proliferation, and causing apoptosis. Our work provides novel mechanistic insights into the action of Hh inhibitors in cancer cells.Item Targeting Heme Function and Mitochondrial Respiration in Non-Small Cell Lung Cancer(2018-12) Kalainayakan, Sarada Preeta; 0000-0003-4332-6425 (Kalainayakan, SP); Zhang, LiEach year lung cancer causes more morbidities than cancers of colon, breast, and prostate combined. The American Cancer Society estimates that lung cancer will claim about 150,000 lives in 2018. Conventional and targeted therapies are reported to have reached the plateau in effectively improving the survival of lung cancer patients. Despite the advent of advanced therapies like immunotherapy, 5-year survival rates remain abysmal at 30% and 10% for Stage III and Stage IV respectively. Therefore, it is imperative to explore different strategies to effectively treat lung cancer and improve survival outcomes. There are two major histological types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for about 85% of the lung cancer cases. Several studies demonstrate that enhanced mitochondrial respiration or oxidative phosphorylation (OXPHOS) is a key feature of NSCLC. Therefore, targeting OXPHOS could be an effective strategy for intervention in NSCLC. Previous studies from our lab showed that hedgehog pathway antagonist, cyclopamine tartrate (CycT), significantly reduced OXPHOS and proliferation in NSCLC cell lines. However, in vitro models do not offer reliable evidence of therapeutic efficacy, thereby, necessitating studies on in vivo models. Previous studies from our lab demonstrated intensified heme uptake and synthesis in NSCLC cell lines compared to normal cell line. Since heme is a central factor in oxygen consumption, this study also probes the effect of CycT on heme metabolism. The objective of this study is two-fold: (i) to test the efficacy of targeting OXPHOS in NSCLC in vivo, and (ii) to investigate the therapeutic efficacy and the mechanism of action of CycT in growth and progression of NSCLC in vivo. We utilized subcutaneous and lung orthotopic xenografts of NSCLC cell lines with luciferase to track the growth and progression of NSCLC in immunodeficient mouse model, NOD/SCID (Non-obese diabetic/ severe combined immunodeficiency), via bioluminescence imaging. The lung tissues of the mice were probed using immunohistochemistry to discern the mechanisms of action. We found that CycT effectively hampered the growth and progression of subcutaneous and lung orthotopic xenografts of NSCLC cell lines. CycT significantly reduced proteins involved in heme metabolism and OXPHOS in addition to other pro-oncogenic hemoproteins and regulators of OXPHOS. In vitro studies demonstrated that the effects of CycT on heme metabolism and OXPHOS are independent of its antagonist properties on the hedgehog pathway. The significance of this study is that it shows that CycT acts via diminishing heme metabolism, hemoproteins involved in oxygen consumption, and oxygen consumption in NSCLC. This is the first study to demonstrate the effect of CycT on heme metabolism and OXPOS in vivo. This novel mechanism of action of CycT is independent of its previously known antagonistic effect on hedgehog signaling. This study demonstrates that CycT has the potential to be an effective therapeutic agent in treating NSCLC. This study provides compelling evidence to further assess the feasibility of using CycT in treating NSCLC.Item The Vascular Disrupting Agent Combretastatin A-4 Phosphate Causes Prolonged Elevation of Proteins Involved in Heme Flux and Function in Resistant Tumor Cells(Impact Journals LLC, 2018-10-22) Dey, Sanchareeka; Kumari, Sharda; Kalainayakan, Sarada Preeta; Campbell, James,,III; Ghosh, Poorva; Zhou, Heling; FitzGerald, Keely E.; Li, Maoping; Mason, Ralph P.; Zhang, Li; Liu, Li; Dey, Sanchareeka; Kalainayakan, Sarada Preeta; Ghosh, Poorva; FitzGerald, Keely E.; Zhang, LiVascular disrupting agents (VDAs) represent a promising class of anti-cancer drugs for solid tumor treatment. Here, we aim to better understand the mechanisms underlying tumor reccurrence and treatment resistance following the administration of a VDA, combretastatin A-4 phosphate (CA4P). Firstly, we used photoacoustic tomography to noninvasively map the effect of CA4P on blood oxygen levels throughout subcutaneous non-small cell lung cancer (NSCLC) tumors in mice. We found that the oxygenation of peripheral tumor vessels was significantly decreased at 1 and 3 hours post-CA4P treatment. The oxygenation of the tumor core reduced significantly at 1 and 3 hours, and reached anoxia after 24 hours. Secondly, we examined the effect of CA4P on the levels of proteins involved in heme flux and function, which are elevated in lung tumors. Using immunohistochemistry, we found that CA4P substantially enhanced the levels of enzymes involved in heme biosynthesis, uptake, and degradation, as well as oxygen-utilizing hemoproteins. Furthermore, measurements of markers of mitochondrial function suggest that CA4P did not diminish mitochondrial function in resistant tumor cells. These results suggest that elevated levels of heme flux and function contribute to tumor regrowth and treatment resistance post-VDA administration.