withanolides and Carcinoma--Renal-Cell

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Biological Products; Carcinoma, Renal Cell; Cell Cycle Proteins; Cell Proliferation; Drug Therapy, Combination; Female; Humans; Immunotherapy; Interferon Inducers; Kidney Neoplasms; Male; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Poly I-C; Transcription Factors; Tumor Cells, Cultured; Withanolides; Xenograft Model Antitumor Assays

Renal cell carcinoma (RCC) is a cancer with poor prognosis, and the 5-year survival rate of patients with metastatic RCC is 5-10%. Consequently, treatment of metastatic RCC represents an unmet clinical need. Screening of a 50 000-member library of natural and synthetic compounds for sensitizers of RCC cells to TRAIL-mediated apoptosis led to identification of the 17β-hydroxywithanolide (17-BHW), withanolide E (1), as a promising lead. To explore structure-activity relationships, we obtained natural and semisynthetic withanolides 1, 2a, 2c, and 3-36 and compared their ability to sensitize TRAIL-mediated apoptosis in a panel of renal carcinoma cells. Our findings revealed that 17-BHWs with a α-oriented side chain are superior to known TRAIL-sensitizing withanolides belonging to withaferin A class with a β-oriented side chain and demonstrated that the 17-BHW scaffold can be modified to enhance sensitization of RCCs to TRAIL-mediated apoptosis, thereby assisting development of natural-product-inspired drugs to treat metastatic RCC.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Renal Cell; Cell Line, Tumor; Ergosterol; Humans; Kidney; Kidney Neoplasms; Structure-Activity Relationship; TNF-Related Apoptosis-Inducing Ligand; Withania; Withanolides

We have purified physapubescin, a predominant steroidal lactone, from medicinal plant Physalis pubescens L., commonly named as "hairy groundcherry" in English and "Deng-Long-Cao" in Chinese. Von Hippel-Lindau (VHL)-null 786-O, RCC4 and A498 Renal Cell Carcinoma (RCC) cell lines expressing high levels of Hypoxia Inducible Factor (HIF)-2α are more sensitive to physapubescin-mediated apoptosis and growth inhibitory effect than VHL wild-type Caki-2 and ACHN RCC cell lines. Restoration of VHL in RCC4 cells attenuated the growth inhibitory effect of physapubescin. Physapubescin decreases the expression of HIF-2α and increases the expression of CCAAT/enhancer-binding protein homologus protein (CHOP), which leads to up-regulation of death receptor 5 (DR5), activation of caspase-8 and -3, cleavage of poly (ADP-Ribose) polymerase (PARP) and apoptosis. Under hypoxia conditions, the apoptotic and growth inhibitory effects of physapubescin are further enhanced. Additionally, physapubescin synergizes with TNF-related apoptosis-inducing ligand (TRAIL) for markedly enhanced induction of apoptosis in VHL-null 786-O cells but not in VHL wild-type Caki-2 cells. Physapubescin significantly inhibited in vivo angiogenesis in the 786-O xenograft. Physapubescin as a novel agent for elimination of VHL-null RCC cells via apoptosis is warranted for further investigation.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Carcinoma, Renal Cell; Caspase 3; Caspase 8; Cell Line, Tumor; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Kidney Neoplasms; Mice; Mice, Nude; Neovascularization, Pathologic; Physalis; Poly(ADP-ribose) Polymerases; Receptors, TNF-Related Apoptosis-Inducing Ligand; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Transcription Factor CHOP; Tumor Burden; Von Hippel-Lindau Tumor Suppressor Protein; Withanolides; Xenograft Model Antitumor Assays

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects.

Topics: Animals; Apoptosis; Blotting, Western; Carcinoma, Renal Cell; Cell Line, Tumor; Endoplasmic Reticulum Stress; Humans; Immunoprecipitation; Mice, Inbred BALB C; Reactive Oxygen Species; TNF-Related Apoptosis-Inducing Ligand; Withanolides

The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) results in cellular stress that initiates a specialized response designated as the unfolded protein response. ER stress has been implicated in a variety of common diseases, such as diabetes, ischemia and neurodegenerative disorders. Withaferin A, a major chemical constituent of Withania somnifera, has been reported to inhibit tumor cell growth. We show that withaferin A induced a dose-dependent apoptotic cell death in several types of human cancer cells, as measured by FACS analysis and PARP cleavage. Treatment of Caki cells with withaferin A induced a number of signature ER stress markers, including phosphorylation of eukaryotic initiation factor-2α (eIF-2 α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78. In addition, withaferin A caused up-regulation of CAAT/enhancer-binding protein-homologous protein (CHOP), suggesting the induction of ER stress. Pretreatment with N-acetyl cysteine (NAC) significantly inhibited withaferin A-mediated ER stress proteins and cell death, suggesting that reactive oxygen species (ROS) mediate withaferin A-induced ER stress. Furthermore, CHOP siRNA or inhibition of caspase-4 activity attenuated withaferin A-induced apoptosis. Taken together, the present study provides strong evidence supporting an important role of the ER stress response in mediating withaferin A-induced apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Renal Cell; Cell Nucleus; Cell Proliferation; Cell Survival; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Endoplasmic Reticulum, Rough; Gene Silencing; HT29 Cells; Humans; Kidney Neoplasms; Oxidative Stress; Regulatory Factor X Transcription Factors; RNA, Messenger; RNA, Small Interfering; Transcription Factor CHOP; Transcription Factors; Withanolides; X-Box Binding Protein 1