T.R. Santhoshkumar

Lysosomal destabilization and cathepsin B contributes for cytochrome c release and caspase activation in embelin-induced apoptosis†

XIAP is an important antiapoptotic protein capable of conferring resistance to cancer cells. Embelin, the small molecular inhibitor of XIAP, possesses wide spectrum of biological activities with strong inhibition of nuclear factor kappa B and downstream antiapoptotic genes. However, the mechanism of its cell death induction is not known. Our studies using colon cancer cells lacking p53 and Bax suggest that both lysosomes and mitochondria are prominent targets of embelin‐induced cell death. Embelin induced cell‐cycle arrest in G1 phase through p21, downstream of p53. In the absence of p21, the cells are sensitized to death in a Bax‐dependent manner. The loss of mitochondrial membrane potential induced by embelin was independent of Bax and p53, but lysosomal integrity loss was strongly influenced by the presence of p53 but not by Bax. Lysosomal role was further substantiated by enhanced cathepsin B activity noticed in embelin‐treated cells. p53‐dependent lysosomal destabilization and cathepsin B activation contribute for increased sensitivity of p21‐deficient cells to embelin with enhanced caspase 9 and caspase 3 activation. Cathepsin B inhibitor reduced cell death and cytochrome c release in embelin‐treated cells indicating lysosomal pathway as the upstream of mitochondrial death signaling. Deficiency of cell‐cycle arrest machinery renders cells more sensitive to embelin with enhanced lysosomal destabilization and caspase processing emphasizing its potential therapeutic importance to address clinical drug resistance.

Essential requirement of cytochrome c release for caspase activation by procaspase-activating compound defined by cellular models

Mitochondrial cytochrome c (cyt. c) release and caspase activation are often impaired in tumors with Bcl-2 overexpression or Bax and Bak-defective status. Direct triggering of cell death downstream of Bax and Bak is an attractive strategy to kill such cancers. Small molecule compounds capable of direct caspase activation appear to be the best mode for killing such tumors. However, there is no precise model to screen such compounds. The currently employed cell-free systems possess the inherent drawback of lacking cellular contents and organelles that operate in integrating cell death signaling. We have developed highly refined cell-based approaches to validate direct caspase activation in cancer cells. Using this approach, we show that PAC-1 (first procaspase-activating compound), the first direct activator of procaspases identified in a cell-free system, in fact requires mitochondrial cyt. c release for triggering caspase activation similar to other antitumor agents. It can induce significant caspase activation and cell death in the absence of Bax and Bak, and in cells overexpressing Bcl-2 and Bcl-xL. This study for the first time defines precise criteria for the validation of direct caspase-activating compounds using specialized cellular models that is expected to accelerate the discovery of potential direct caspase activators.

Metastasis-associated Protein 1 Drives Tumor Cell Migration and Invasion through Transcriptional Repression of RING Finger Protein 144A

Background: The mechanistic role of MTA1 in tumor aggressiveness is yet to be deciphered. Results: RNF144A is a direct target of transcriptional repression by MTA1 and inhibits migration and invasion. Conclusion: Transcriptional repression of RNF144A by MTA1 confers a migratory and invasive phenotype of cancer cells. Significance: This study provides novel mechanistic insights into regulation of tumor progression by MTA1. Metastasis-associated protein 1 (MTA1), a component of the nucleosome-remodeling and histone deacetylase complex, is widely up-regulated in human cancers and significantly correlated with tumor invasion and metastasis, but the mechanisms involved remain largely unknown. Here, we report that MTA1 transcriptionally represses the expression of RING finger protein 144A (RNF144A), an uncharacterized gene whose protein product possesses potential E3 ubiquitin ligase activity, by recruiting the histone deacetylase 2 (HDAC2) and CCAAT/enhancer-binding protein α (c/EBPα) co-repressor complex onto human RNF144A promoter. Furthermore, an inverse correlation between the expression levels of MTA1 and RNF144A was demonstrated in publicly available breast cancer microarray datasets and the MCF10 breast cancer progression model system. To address functional aspects of MTA1 regulation of RNF144A, we demonstrate that RNF144A is a novel suppressor of cancer migration and invasion, two requisite steps of metastasis in vivo, and knockdown of endogenous RNF144A by small interfering RNAs accelerates the migration and invasion of MTA1-overexpressing cells. These results suggest that RNF144A is partially responsible for MTA1-mediated migration and invasion and that MTA1 overexpression in highly metastatic cancer cells drives cell migration and invasion by, at least in part, interfering with the suppressive function of RNF144A through transcriptional repression of RNF144A expression. Together, these findings provide novel mechanistic insights into regulation of tumor progression and metastasis by MTA1 and highlight a previously unrecognized role of RNF144A in MTA1-driven cancer cell migration and invasion.

ERO1α-dependent endoplasmic reticulum-mitochondrial calcium flux contributes to ER stress and mitochondrial permeabilization by procaspase-activating compound-1 (PAC-1).

Procaspase-activating compound-1 (PAC-1) is the first direct caspase-activating compound discovered; using an in vitro cell-free system of caspase activation. Subsequently, this compound was shown to induce apoptosis in a variety of cancer cells with promising in vivo antitumor activity in canine lymphoma model. Recently, we have reported its ability to kill drug-resistant, Bcl-2/Bcl-xL overexpressing and Bax/Bak-deficient cells despite the essential requirement of mitochondrial cytochrome c (cyt. c) release for caspase activation, indicating that the key molecular targets of PAC-1 in cancer cells are yet to be identified. Here, we have identified Ero1α-dependent endoplasmic reticulum (ER) calcium leakage to mitochondria through mitochondria-associated ER membranes (MAM) and ER luminal hyper-oxidation as the critical events of PAC-1-mediated cell death. PAC-1 treatment upregulated Ero1α in multiple cell lines, whereas silencing of Ero1α significantly inhibited calcium release from ER and cell death. Loss of ER calcium and hyper-oxidation of ER lumen by Ero1α collectively triggered ER stress. Upregulation of GRP78 and splicing of X-box-binding protein 1 (XBP1) mRNA in multiple cancer cells suggested ER stress as the general event triggered by PAC-1. XBP1 mRNA splicing and GRP78 upregulation confirmed ER stress even in Bax/Bak double knockout and PAC-1-resistant Apaf-1-knockout cells, indicating an induction of ER stress-mediated mitochondrial apoptosis by PAC-1. Furthermore, we identified BH3-only protein p53 upregulated modulator of apoptosis (PUMA) as the key molecular link that orchestrates overwhelmed ER stress to mitochondria-mediated apoptosis, involving mitochondrial reactive oxygen species, in a p53-independent manner. Silencing of PUMA in cancer cells effectively reduced cyt. c release and cell death by PAC-1.

Bax deficiency mediated drug resistance can be reversed by endoplasmic reticulum stress induced death signaling

Tumors often acquire drug resistance due to functional loss of pro apoptotic gene Bax, a critical and essential component of cell death rendering them insensitive to most anti-tumor agents. Compounds that can induce Bax independent apoptotic cell death are expected to overcome such drug resistance. We have employed a live cell based screening platform to identify potential compounds that can induce programmed cell death in Bax deficiency. Release of cytochrome C from mitochondria into the cytosol is a decisive initial event required for the caspase dependent cell death. We have engineered both wild type and Bax knock out colon cancer cells stably expressing cytochrome C with EGFP fusion protein to identify compounds that can trigger cytochrome C release in both cells with equal efficiency. In the fluorescent translocation assay, most of the drugs tested failed to induce cytochrome C release in Bax deficient cells validating the sensitivity of the assay. This study identified five lead compounds such as thapsigargin, tunicamycine, MG132, kaempferol and camptothecin that could induce cytochrome C release in both wild type and Bax deficient cells with equal potency. All the positive hits induced ER stress signaling as evidenced by up-regulation of Grp78. Studies with a Bak deficient cells indicate that Bak deficiency confers protection to cells from ER stress through autophagy. Further studies revealed that ER stress inducing agents are capable of triggering classical mitochondrial apoptotic cell death through the conformational activation of Bak, substantiating the potential of this pathway in designing drugs against Bax deficiency mediated drug resistance.

Identification of heat shock protein 90 inhibitors to sensitize drug resistant side population tumor cells using a cell based assay platform.

Current cancer therapeutics are identified based on initial tumor regression screens that mostly kill differentiated tumor cells, sparing the rare cancer stem cells (CSCs). Being rare and difficult to characterize, it remains a challenge to identify compounds active against them. Side population (SP) cells identified in multiple cancer cell line panels expressing mitochondrial Cytochrome C-EGFP were evaluated for identifying possible drug candidates utilizing high-throughput imaging. We identified heat shock protein 90 inhibitors as potential agents to sensitize SP cells to anticancer drugs. Hsp90 inhibitors induced down regulation of Akt leading to proteasomal degradation of survivin and consequent mitochondrial apoptosis. A successful screening platform for identifying compounds targeting drug resistant side population cells was developed.

Novel BCL2 inhibitor, Disarib Induces Apoptosis by Disruption of BCL2-BAK Interaction.

ABSTRACT Apoptosis is a highly regulated pathway of programmed cell death relying on the fine balance between pro and antiapoptotic binding partners. Overexpression of the antiapoptotic protein BCL2 in several cancers makes it an ideal target for chemotherapy, with minimum side effects. In one of our previous studies, we designed, synthesized and characterized Disarib, a BCL2‐specific small molecule inhibitor. Interestingly, Disarib showed a novel mode of BCL2 inhibition, by predominantly binding to its BH1 domain, as compared to the BH3‐specific action of other known BCL2 inhibitors. Here, we investigate the mechanism by which Disarib induces cell death, upon binding to BCL2. We find that Disarib specifically disrupted the BCL2‐BAK interaction, but not that of BCL2‐BAX or other members of the proapoptotic family such as PUMA and BIM, in vitro. Biochemical and biophysical studies demonstrate Disarib‐induced inhibition of BCL2‐BAK interaction with a Ki of 12.76 nM. Genetic knockout cells of BAK/BAX and double knockout (DKO) cells confirmed a BAK‐specific action of Disarib, thereby facilitating apoptosis. Importantly, intracellular FRET in BAK/BAX single and double knockout cells demonstrated BCL2‐BAK disruption, and activation of intrinsic pathway of apoptosis upon Disarib treatment. Thus, we report a unique mechanism of action of a BCL2 inhibitor, Disarib, by specifically targeting the interaction of BCL2‐BAK, while sparing that of other proapoptotic binding partners.

A high-throughput image-based screen for the identification of Bax/Bak-independent caspase activators against drug-resistant cancer cells

Despite the use of new generation target specific drugs or combination treatments, drug-resistance caused by defective apoptosis signaling remains a major challenge in cancer treatment. A common apoptotic defect in drug-resistant tumor is the failure of cancer cells to undergo Bax/Bak-dependent mitochondrial permeabilization due to impaired signaling of Bcl-2 family proteins. Therefore, Bax and Bak-independent caspase-activating compounds appear to be effective in killing such tumor cells. An image-based cellular platform of caspase sensors in Bax and Bak deficient background allowed us to identify several potential Bax/Bak-independent caspase-activating compounds from a limited high-throughput compound screening. FRET-based caspase sensor probe targeted at the nucleus enabled accurate and automated segmentation, yielding a Z-value of 0.72. Some of the positive hits showed promising activity against drug-resistant human cancer cells expressing high levels of Bcl-2 or Bcl-xL. Using this approach, we describe thiolutin, CD437 and TPEN as the most potentially valuable drug candidates for addressing drug-resistance caused by aberrant expression of Bcl-2 family proteins in tumor cells. The screen also enables the quantification of multiparameter apoptotic events along with caspase activation in HTS manner in live mode, allowing characterization of non-classical apoptosis signaling.

Endoplasmic Reticulum-Targeted Bcl-2 Inhibitable Mitochondrial Fragmentation Initiates ER Stress-Induced Cell Death

Endoplasmic reticulum (ER) plays a key role in the maintenance of properly folded proteins, intracellular calcium, modification and trafficking of secretary proteins, etc. Any functional disturbance in ER triggers programmed cell death if reestablishment of homeostasis is failed. Several studies suggest toward a unique ER-centered apoptotic signaling separate from the Bax and Bak-dependent intrinsic apoptosis signaling during ER stress. Here, we show that significant loss of mitochondrial network and fragmentation before cytochrome c release during ER stress initiates the death signaling. The use of Bax-EGFP cells expressing MitoDsRed established that the mitochondrial fragmentation event during ER stress is upstream of Bax activation and its translocation to mitochondria. Caspase inhibitors failed to alter the mitochondrial fragmentation induced by multiple ER stress inducing agents. However, Bcl-2 overexpression specifically at ER significantly prevented early mitochondrial fragmentation and cell death induced by ER stress than the wild type Bcl-2 expressing cells. Our studies suggest that an early caspase-independent mitochondrial fragmentation as an initiating event during ER stress-induced cell death that is under the regulation of Bcl-2 resident at the ER.

A novel inhibitor of BCL2, Disarib abrogates tumor growth while sparing platelets, by activating intrinsic pathway of apoptosis

Antiapoptotic protein BCL2, serves as an excellent target for anticancer therapy owing to its increased level in cancers. Previously, we have described characterization of a novel BCL2 inhibitor, Disarib, which showed selective cytotoxicity in BCL2 high cancer cells and CLL patient cells. Here, we have investigated the mechanism of Disarib-induced cytotoxicity, and compared its efficacy with a well-established BCL2 inhibitor, ABT199. We show that Disarib administration caused tumor regression in mouse allograft and xenograft models, exhibited platelet sparing property and did not exhibit significant side effects. Importantly, comparison between Disarib and ABT199, revealed higher efficacy for Disarib in mouse tumor model and cancer cell lines. Disarib induced cell death by activating intrinsic apoptotic pathway. Interestingly, Disarib showed synergism with paclitaxel, suggesting its potential for combination therapy. Thus, we provide mechanistic insights into the cell death pathways induced by Disarib, report that Disarib exhibited better effect than currently used ABT199 and demonstrate its combinatorial potential with paclitaxel.