Dhyan Chandra - Academia.edu (original) (raw)
Papers by Dhyan Chandra
Journal of Hematology & Oncology
Mitochondria are essential for tumor growth and progression. However, the heavy demand for mitoch... more Mitochondria are essential for tumor growth and progression. However, the heavy demand for mitochondrial activity in cancer leads to increased production of mitochondrial reactive oxygen species (mtROS), accumulation of mutations in mitochondrial DNA, and development of mitochondrial dysfunction. If left unchecked, excessive mtROS can damage and unfold proteins in the mitochondria to an extent that becomes lethal to the tumor. Cellular systems have evolved to combat mtROS and alleviate mitochondrial stress through a quality control mechanism called the mitochondrial unfolded protein response (UPRmt). The UPRmt system is composed of chaperones and proteases, which promote protein folding or eliminate mitochondrial proteins damaged by mtROS, respectively. UPRmt is conserved and activated in cancer in response to mitochondrial stress to maintain mitochondrial integrity and support tumor growth. In this review, we discuss how mitochondria become dysfunctional in cancer and highlight the...
Nucleotide regulates apoptosome-mediated caspase activation
<p><b>A</b>, Summary of cell-free apoptosome reconstitution assays using recomb... more <p><b>A</b>, Summary of cell-free apoptosome reconstitution assays using recombinant proteins, or fresh or stored cytosols. Dashed arrows in ‘Stored cytosol’ indicate attenuated caspase activation. <b>B,</b> Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016379#s3" target="_blank">Discussion</a> for details. Casp-9, caspase-9.</p
Food and Chemical Toxicology, 2001
There is a growing interest in understanding the biological effect of time-tested folk medicinal ... more There is a growing interest in understanding the biological effect of time-tested folk medicinal plants. In the present work, the effect of dietary administration of fenugreek seeds on the glyoxalase system and antioxidant enzymes as well as levels of glutathione (GSH) have been examined in different tissues of mice. Swiss albino mice were fed with diet containing powdered fenugreek for 4 weeks. The mode and magnitude of effect was found to depend on the dose of fenugreek and type of tissue. The diet containing 1 and 2% of fenugreek enhanced the specific activity of gly I, whereas 5 and 10% inhibited its activity in the liver. However, on the other hand, the specific activity of gly II decreased continuously with dose. Dietary administration of fenugreek seeds resulted in an increase of GSH levels and the glutathione S-transferase (GST) activity in the liver. Fenugreek (1 and 2%) showed no appreciable change in superoxide dismutase (SOD) and catalase. However, 5 and 10% doses of fenugreek supplementation inhibited their activities. In the case of spleen, a significant change in gly I was observed only at 10% fenugreek. In addition, a significant increase was also observed with GSH levels. Fenugreek treatment up to 5% showed a small but consistent increase in erythrocytes. In the case of GST, the activity enhanced with dose. Significant elevation of catalase activity was observed with a 10% dose of fenugreek. The significance and relevance of these findings are suggested in relation to mechanistic aspects.
Journal of Clinical Investigation
Mitochondrial Regulation of Inflammation in Cancer
Physiology in Health and Disease, 2021
Small Molecule MMRi62 Induces Ferroptosis and Inhibits Metastasis in Pancreatic Cancer via Degradation of Ferritin Heavy Chain and Mutant p53
Molecular Cancer Therapeutics, 2022
High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenoc... more High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenocarcinoma (PDAC). TP53 mutation not only renders PDAC resistance to chemotherapies but also drives PDAC invasiveness. Therapies targeting activating mutant KRAS are not available and the outcomes of current PDAC treatment are extremely poor. Here, we report that MMRi62, initially identified as an MDM2-MDM4–targeting small molecule with p53-independent pro-apoptotic activity, shows anti-PDAC activity in vitro and in vivo. We show that MMRi62 inhibits proliferation, clonogenic, and spheroid growth of PDAC cells by induction of cell death. MMRi62-induced cell death in PDAC is characteristic of ferroptosis that is associated with increased autophagy, increased reactive oxygen species, and lysosomal degradation of NCOA4 and ferritin heavy chain (FTH1). In addition to induced degradation of FTH1, MMRi62 also induces proteasomal degradation of mutant p53. Interestingly, MMRi62-induced ferroptosi...
Radiation Therapy-Induced Metastasis and Secondary Malignancy
Radiation therapy (RT) is a common anticancer regimen for the management of many types of human m... more Radiation therapy (RT) is a common anticancer regimen for the management of many types of human malignancies. However, the development of secondary malignant neoplasms (SMNs) after RT remains a major problem in the clinic, and is mainly due to the increase in number of cancer survivors. SMNs contribute to the major problem for patients because 1 in 12 cancer survivors develop SMN. The majority of these patients succumb to the SMN, thereby decreasing overall survival for cancer patients. Therefore, it is important to understand how SMNs develop and ways to counteract this serious health concern among cancer patients. Although multiple mechanisms may characterize SMN development and prognosis, this review will specifically discuss how RT induces metastasis thereby leading to SMN development and therapeutic approaches to overcome this problem.
Targeting the mitochondrial unfolded protein response in cancer: opportunities and challenges
Trends in Cancer, 2021
Increasing evidence indicates that a mitochondria-specific stress response referred to as the ... more Increasing evidence indicates that a mitochondria-specific stress response referred to as the 'mitochondrial unfolded protein response' (UPRmt) is activated to maintain mitochondrial integrity and support tumor growth. In this forum article, we discuss the recent advances and current challenges in therapeutically targeting UPRmt in cancer.
Effects of anti-cancer drug doxorubicin on endogenous biomarkers NAD(P)H, FAD and Trp in prostate cancer cells: a FLIM Study
SPIE Proceedings, 2017
Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to identify changes in metabolic acti... more Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to identify changes in metabolic activity during cancer progression and upon anti-cancer drug treatment. Prostate cancer (PCa) is one of the leading cancers in men in the USA. This research focusses on understanding the lifetime changes of endogenous biomarkers: NAD(P)H, FAD and Trp in LNCaP cells upon treatment with doxorubicin using our 3-channel FLIM approach. The LNCaP cells were treated with doxorubicin for 24hr. Images using FLIM of LNCaP control and treated cells were acquired on Zeiss 780 multiphoton confocal microscope coupled with B and H TCSPC FLIM board. After FLIM data fitting and processing we observed increase in the mean fluorescence lifetime of Trp, NAD(P)H and FAD with doxorubicin treatment. Additionally, we saw reduction in the NAD(P)H/FAD redox ratio with doxorubicin treatment. Our results identify the changes in the lifetime of these endogenous biomarkers and in the cellular redox state as a metabolic response with doxorubicin treatment in prostate cancer cells.
Investigation of prostate cancer cells using NADH and tryptophan as biomarker: multiphoton FLIM-FRET microscopy
Multiphoton Microscopy in the Biomedical Sciences XVI, 2016
Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. ... more Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. Prostate cancer is one of the leading cancers in men in the USA. This research focuses on FLIM measurements of NAD(P)H and Tryptophan, used as biomarkers to understand the metabolic activity in prostate cancer cells. Two prostate cancers and one normal cell line were used for live-cell FLIM measurements on Zeiss780 2P confocal microscope with SPCM FLIM board. Glucose uptake and glycolysis proceeds about ten times faster in cancer than in non-cancerous tissues. Therefore, we assessed the glycolytic activity in the prostate cancer in comparison to the normal cells upon glucose stimulation by analyzing the NAD(P)H and Trp lifetime distribution and efficiency of energy transfer (E%). Furthermore, we treated the prostate cancer cells with 1μM Doxorubicin, a commonly used anti-cancer chemotherapeutic. Increase in NADH a2%, an indicator of increased glycolysis and increased E% between Trp and NAD(P)H were seen upon glucose stimulation for 30min. The magnitude of shift to the right for NAD(P)H a2% and E% distribution was higher in prostate cancer versus the normal cells. Upon treatment with Doxorubicin decrease in cellular metabolism was seen at 15 and 30 minutes. The histogram for NAD(P)H a2% post-treatment for prostate cancer cells showed a left shift compared to the untreated control suggesting decrease in glycolysis and metabolic activity opposite to what was observed after glucose stimulation. Hence, NAD(P)H and Trp lifetimes can be used biomarkers to understand metabolic activity in prostate cancer and upon chemotherapeutic interventions.
Cell death & disease, Jan 5, 2015
Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt... more Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase ...
Cell death & disease, Jan 9, 2014
Apoptosis is a dynamic process regulated by mitochondrion critical for cellular respiration and s... more Apoptosis is a dynamic process regulated by mitochondrion critical for cellular respiration and survival. Execution of apoptosis is mediated by multiple protein signaling events at mitochondria. Initiation and progression of apoptosis require numerous apoptogenic factors that are either released from or sequestered in mitochondria, which may transform the biomolecular makeup of the organelle. In this communication, using Raman microspectroscopy, we demonstrate that transformation in biomolecular composition of mitochondrion may be used as apoptosis marker in an individual cell. For the first time, we show that significant changes occur in the concentrations of RNA, DNA, protein, and lipid constituents of mitochondria during apoptosis. The structural analysis of proteins on mitochondria demonstrated a decrease in α-helix secondary structure content, and an increase in the levels of random coils and β-sheets on mitochondria. This may represent an additional hallmark of apoptosis. Stri...
Identification and Characterization of Bim, a Novel Proapoptotic BH3-only Splice Variant of Bim1
BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In ... more BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In this study, a novel human BH3-only protein, Bcl-2-interacting mediator (Bim), was identified during our study of regulation of prostate cancer cell death by Bcl-2 family proteins. Bim shares the highest amino acid sequence homology to BimEL and BimL, two proapoptotic BH3-only Bcl-2 proteins derived
Identification and Characterization of Bim, a Novel Proapoptotic BH3-only Splice Variant of Bim1
BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In ... more BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In this study, a novel human BH3-only protein, Bcl-2-interacting mediator (Bim), was identified during our study of regulation of prostate cancer cell death by Bcl-2 family proteins. Bim shares the highest amino acid sequence homology to BimEL and BimL, two proapoptotic BH3-only Bcl-2 proteins derived
PLoS ONE, 2011
In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol trigg... more In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.
PLoS ONE, 2014
Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for th... more Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ,99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/ NanogP8 is ,35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ,22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ,28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.
Cell, 2006
Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apop... more Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death.
Carcinogenesis, 2012
Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extrac... more Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and-9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neeminduced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neeminduced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.
Identification and Characterization of Bimγ, a Novel Proapoptotic BH3-Only Splice Variant of Bim
Cancer research, 2002
BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essentialrole in apoptosis. In t... more BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essentialrole in apoptosis. In this study, a novel human BH3-only protein, Bcl-2-interacting mediator (Bim)γ, was identified during our study of regulation of prostate cancer cell death by Bcl-2 family proteins. Bimγ ...
PLoS ONE, 2013
Reduction or depletion of mitochondrial DNA (mtDNA) has been associated with cancer progression. ... more Reduction or depletion of mitochondrial DNA (mtDNA) has been associated with cancer progression. Although imbalanced mtDNA content is known to occur in prostate cancer, differences in mtDNA content between African American (AA) and Caucasian American (CA) men are not defined. We provide the first evidence that tumors in AA men possess reduced level of mtDNA compared to CA men. The median tumor mtDNA content was reduced in AA men. mtDNA content was also reduced in normal prostate tissues of AA men compared to CA men, suggesting a possible predisposition to cancer in AA men. mtDNA content was also reduced in benign prostatic hyperplasia (BPH) tissue from AA men. Tumor and BPH tissues from patients $60 years of age possess reduced mtDNA content compared to patients ,60 years of age. In addition, mtDNA content was higher in normal tissues from patients with malignant T3 stage disease compared to patients with T2 stage disease. mtDNA levels in matched normal prostate tissues were nearly doubled in Gleason grade of .7 compared to #7, whereas reduced mtDNA content was observed in tumors of Gleason grade .7 compared to #7. Together, our data suggest that AA men possess lower mtDNA levels in normal and tumor tissues compared to CA men, which could contribute to higher risk and more aggressive prostate cancer in AA men.
Journal of Hematology & Oncology
Mitochondria are essential for tumor growth and progression. However, the heavy demand for mitoch... more Mitochondria are essential for tumor growth and progression. However, the heavy demand for mitochondrial activity in cancer leads to increased production of mitochondrial reactive oxygen species (mtROS), accumulation of mutations in mitochondrial DNA, and development of mitochondrial dysfunction. If left unchecked, excessive mtROS can damage and unfold proteins in the mitochondria to an extent that becomes lethal to the tumor. Cellular systems have evolved to combat mtROS and alleviate mitochondrial stress through a quality control mechanism called the mitochondrial unfolded protein response (UPRmt). The UPRmt system is composed of chaperones and proteases, which promote protein folding or eliminate mitochondrial proteins damaged by mtROS, respectively. UPRmt is conserved and activated in cancer in response to mitochondrial stress to maintain mitochondrial integrity and support tumor growth. In this review, we discuss how mitochondria become dysfunctional in cancer and highlight the...
Nucleotide regulates apoptosome-mediated caspase activation
<p><b>A</b>, Summary of cell-free apoptosome reconstitution assays using recomb... more <p><b>A</b>, Summary of cell-free apoptosome reconstitution assays using recombinant proteins, or fresh or stored cytosols. Dashed arrows in ‘Stored cytosol’ indicate attenuated caspase activation. <b>B,</b> Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016379#s3" target="_blank">Discussion</a> for details. Casp-9, caspase-9.</p
Food and Chemical Toxicology, 2001
There is a growing interest in understanding the biological effect of time-tested folk medicinal ... more There is a growing interest in understanding the biological effect of time-tested folk medicinal plants. In the present work, the effect of dietary administration of fenugreek seeds on the glyoxalase system and antioxidant enzymes as well as levels of glutathione (GSH) have been examined in different tissues of mice. Swiss albino mice were fed with diet containing powdered fenugreek for 4 weeks. The mode and magnitude of effect was found to depend on the dose of fenugreek and type of tissue. The diet containing 1 and 2% of fenugreek enhanced the specific activity of gly I, whereas 5 and 10% inhibited its activity in the liver. However, on the other hand, the specific activity of gly II decreased continuously with dose. Dietary administration of fenugreek seeds resulted in an increase of GSH levels and the glutathione S-transferase (GST) activity in the liver. Fenugreek (1 and 2%) showed no appreciable change in superoxide dismutase (SOD) and catalase. However, 5 and 10% doses of fenugreek supplementation inhibited their activities. In the case of spleen, a significant change in gly I was observed only at 10% fenugreek. In addition, a significant increase was also observed with GSH levels. Fenugreek treatment up to 5% showed a small but consistent increase in erythrocytes. In the case of GST, the activity enhanced with dose. Significant elevation of catalase activity was observed with a 10% dose of fenugreek. The significance and relevance of these findings are suggested in relation to mechanistic aspects.
Journal of Clinical Investigation
Mitochondrial Regulation of Inflammation in Cancer
Physiology in Health and Disease, 2021
Small Molecule MMRi62 Induces Ferroptosis and Inhibits Metastasis in Pancreatic Cancer via Degradation of Ferritin Heavy Chain and Mutant p53
Molecular Cancer Therapeutics, 2022
High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenoc... more High frequency of KRAS and TP53 mutations is a unique genetic feature of pancreatic ductal adenocarcinoma (PDAC). TP53 mutation not only renders PDAC resistance to chemotherapies but also drives PDAC invasiveness. Therapies targeting activating mutant KRAS are not available and the outcomes of current PDAC treatment are extremely poor. Here, we report that MMRi62, initially identified as an MDM2-MDM4–targeting small molecule with p53-independent pro-apoptotic activity, shows anti-PDAC activity in vitro and in vivo. We show that MMRi62 inhibits proliferation, clonogenic, and spheroid growth of PDAC cells by induction of cell death. MMRi62-induced cell death in PDAC is characteristic of ferroptosis that is associated with increased autophagy, increased reactive oxygen species, and lysosomal degradation of NCOA4 and ferritin heavy chain (FTH1). In addition to induced degradation of FTH1, MMRi62 also induces proteasomal degradation of mutant p53. Interestingly, MMRi62-induced ferroptosi...
Radiation Therapy-Induced Metastasis and Secondary Malignancy
Radiation therapy (RT) is a common anticancer regimen for the management of many types of human m... more Radiation therapy (RT) is a common anticancer regimen for the management of many types of human malignancies. However, the development of secondary malignant neoplasms (SMNs) after RT remains a major problem in the clinic, and is mainly due to the increase in number of cancer survivors. SMNs contribute to the major problem for patients because 1 in 12 cancer survivors develop SMN. The majority of these patients succumb to the SMN, thereby decreasing overall survival for cancer patients. Therefore, it is important to understand how SMNs develop and ways to counteract this serious health concern among cancer patients. Although multiple mechanisms may characterize SMN development and prognosis, this review will specifically discuss how RT induces metastasis thereby leading to SMN development and therapeutic approaches to overcome this problem.
Targeting the mitochondrial unfolded protein response in cancer: opportunities and challenges
Trends in Cancer, 2021
Increasing evidence indicates that a mitochondria-specific stress response referred to as the ... more Increasing evidence indicates that a mitochondria-specific stress response referred to as the 'mitochondrial unfolded protein response' (UPRmt) is activated to maintain mitochondrial integrity and support tumor growth. In this forum article, we discuss the recent advances and current challenges in therapeutically targeting UPRmt in cancer.
Effects of anti-cancer drug doxorubicin on endogenous biomarkers NAD(P)H, FAD and Trp in prostate cancer cells: a FLIM Study
SPIE Proceedings, 2017
Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to identify changes in metabolic acti... more Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to identify changes in metabolic activity during cancer progression and upon anti-cancer drug treatment. Prostate cancer (PCa) is one of the leading cancers in men in the USA. This research focusses on understanding the lifetime changes of endogenous biomarkers: NAD(P)H, FAD and Trp in LNCaP cells upon treatment with doxorubicin using our 3-channel FLIM approach. The LNCaP cells were treated with doxorubicin for 24hr. Images using FLIM of LNCaP control and treated cells were acquired on Zeiss 780 multiphoton confocal microscope coupled with B and H TCSPC FLIM board. After FLIM data fitting and processing we observed increase in the mean fluorescence lifetime of Trp, NAD(P)H and FAD with doxorubicin treatment. Additionally, we saw reduction in the NAD(P)H/FAD redox ratio with doxorubicin treatment. Our results identify the changes in the lifetime of these endogenous biomarkers and in the cellular redox state as a metabolic response with doxorubicin treatment in prostate cancer cells.
Investigation of prostate cancer cells using NADH and tryptophan as biomarker: multiphoton FLIM-FRET microscopy
Multiphoton Microscopy in the Biomedical Sciences XVI, 2016
Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. ... more Fluorescence Lifetime Imaging (FLIM) can be used to understand the metabolic activity in cancer. Prostate cancer is one of the leading cancers in men in the USA. This research focuses on FLIM measurements of NAD(P)H and Tryptophan, used as biomarkers to understand the metabolic activity in prostate cancer cells. Two prostate cancers and one normal cell line were used for live-cell FLIM measurements on Zeiss780 2P confocal microscope with SPCM FLIM board. Glucose uptake and glycolysis proceeds about ten times faster in cancer than in non-cancerous tissues. Therefore, we assessed the glycolytic activity in the prostate cancer in comparison to the normal cells upon glucose stimulation by analyzing the NAD(P)H and Trp lifetime distribution and efficiency of energy transfer (E%). Furthermore, we treated the prostate cancer cells with 1μM Doxorubicin, a commonly used anti-cancer chemotherapeutic. Increase in NADH a2%, an indicator of increased glycolysis and increased E% between Trp and NAD(P)H were seen upon glucose stimulation for 30min. The magnitude of shift to the right for NAD(P)H a2% and E% distribution was higher in prostate cancer versus the normal cells. Upon treatment with Doxorubicin decrease in cellular metabolism was seen at 15 and 30 minutes. The histogram for NAD(P)H a2% post-treatment for prostate cancer cells showed a left shift compared to the untreated control suggesting decrease in glycolysis and metabolic activity opposite to what was observed after glucose stimulation. Hence, NAD(P)H and Trp lifetimes can be used biomarkers to understand metabolic activity in prostate cancer and upon chemotherapeutic interventions.
Cell death & disease, Jan 5, 2015
Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt... more Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase ...
Cell death & disease, Jan 9, 2014
Apoptosis is a dynamic process regulated by mitochondrion critical for cellular respiration and s... more Apoptosis is a dynamic process regulated by mitochondrion critical for cellular respiration and survival. Execution of apoptosis is mediated by multiple protein signaling events at mitochondria. Initiation and progression of apoptosis require numerous apoptogenic factors that are either released from or sequestered in mitochondria, which may transform the biomolecular makeup of the organelle. In this communication, using Raman microspectroscopy, we demonstrate that transformation in biomolecular composition of mitochondrion may be used as apoptosis marker in an individual cell. For the first time, we show that significant changes occur in the concentrations of RNA, DNA, protein, and lipid constituents of mitochondria during apoptosis. The structural analysis of proteins on mitochondria demonstrated a decrease in α-helix secondary structure content, and an increase in the levels of random coils and β-sheets on mitochondria. This may represent an additional hallmark of apoptosis. Stri...
Identification and Characterization of Bim, a Novel Proapoptotic BH3-only Splice Variant of Bim1
BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In ... more BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In this study, a novel human BH3-only protein, Bcl-2-interacting mediator (Bim), was identified during our study of regulation of prostate cancer cell death by Bcl-2 family proteins. Bim shares the highest amino acid sequence homology to BimEL and BimL, two proapoptotic BH3-only Bcl-2 proteins derived
Identification and Characterization of Bim, a Novel Proapoptotic BH3-only Splice Variant of Bim1
BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In ... more BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essential role in apoptosis. In this study, a novel human BH3-only protein, Bcl-2-interacting mediator (Bim), was identified during our study of regulation of prostate cancer cell death by Bcl-2 family proteins. Bim shares the highest amino acid sequence homology to BimEL and BimL, two proapoptotic BH3-only Bcl-2 proteins derived
PLoS ONE, 2011
In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol trigg... more In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.
PLoS ONE, 2014
Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for th... more Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ,99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/ NanogP8 is ,35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29-80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ,22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ,28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells.
Cell, 2006
Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apop... more Cytochrome c (CC)-initiated Apaf-1 apoptosome formation represents a key initiating event in apoptosis. This process can be reconstituted in vitro with the addition of CC and ATP or dATP to cell lysates. How physiological levels of nucleotides, normally at high mM concentrations, affect apoptosome activation remains unclear. Here we show that physiological levels of nucleotides inhibit the CC-initiated apoptosome formation and caspase-9 activation by directly binding to CC on several key lysine residues and thus preventing CC interaction with Apaf-1. We show that in various apoptotic systems caspase activation is preceded or accompanied by decreases in overall intracellular NTP pools. Microinjection of nucleotides inhibits whereas experimentally reducing NTP pools enhances both CC and apoptotic stimuli-induced cell death. Our results thus suggest that the intracellular nucleotides represent critical prosurvival factors by functioning as natural inhibitors of apoptosome formation and a barrier that cells must overcome the nucleotide barrier to undergo apoptosis cell death.
Carcinogenesis, 2012
Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extrac... more Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and-9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neeminduced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neeminduced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.
Identification and Characterization of Bimγ, a Novel Proapoptotic BH3-Only Splice Variant of Bim
Cancer research, 2002
BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essentialrole in apoptosis. In t... more BH3 (Bcl-2 homology 3)-only proteins of the Bcl-2 family play an essentialrole in apoptosis. In this study, a novel human BH3-only protein, Bcl-2-interacting mediator (Bim)γ, was identified during our study of regulation of prostate cancer cell death by Bcl-2 family proteins. Bimγ ...
PLoS ONE, 2013
Reduction or depletion of mitochondrial DNA (mtDNA) has been associated with cancer progression. ... more Reduction or depletion of mitochondrial DNA (mtDNA) has been associated with cancer progression. Although imbalanced mtDNA content is known to occur in prostate cancer, differences in mtDNA content between African American (AA) and Caucasian American (CA) men are not defined. We provide the first evidence that tumors in AA men possess reduced level of mtDNA compared to CA men. The median tumor mtDNA content was reduced in AA men. mtDNA content was also reduced in normal prostate tissues of AA men compared to CA men, suggesting a possible predisposition to cancer in AA men. mtDNA content was also reduced in benign prostatic hyperplasia (BPH) tissue from AA men. Tumor and BPH tissues from patients $60 years of age possess reduced mtDNA content compared to patients ,60 years of age. In addition, mtDNA content was higher in normal tissues from patients with malignant T3 stage disease compared to patients with T2 stage disease. mtDNA levels in matched normal prostate tissues were nearly doubled in Gleason grade of .7 compared to #7, whereas reduced mtDNA content was observed in tumors of Gleason grade .7 compared to #7. Together, our data suggest that AA men possess lower mtDNA levels in normal and tumor tissues compared to CA men, which could contribute to higher risk and more aggressive prostate cancer in AA men.