High-content drug screening in zebrafish xenografts reveals high efficacy of dual MCL-1/BCL-XL inhibition against Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue cancer with an urgent need for new therapies to improve disease outcome. To identify effective drugs, phenotypic drug screening has proven to be a powerful method, but achievable throughput in mouse xenografts, the preclinical Ewing sarcoma standard model, is limited. Here, we explored the use of xenografts in zebrafish for high-throughput drug screening to discover new combination therapies for Ewing sarcoma. We subjected xenografts in zebrafish larvae to high-content imaging and subsequent automated tumor size analysis to screen single agents and compound combinations. We identified three drug combinations effective against Ewing sarcoma cells: Irinotecan combined with either an MCL-1 or an BCL-XL inhibitor and in particular dual inhibition of the anti-apoptotic proteins MCL-1 and BCL-XL, which efficiently eradicated tumor cells in zebrafish xenografts. We confirmed enhanced efficacy of dual MCL-1/BCL-XL inhibition compared to single agents in a mouse PDX model. In conclusion, high-content screening of small compounds on Ewing sarcoma zebrafish xenografts identified dual MCL-1/BCL-XL targeting as a specific vulnerability and promising therapeutic strategy for Ewing sarcoma, which warrants further investigation towards clinical application.

Downregulation of Microrna-421 Relieves Cerebral Ischemia/Reperfusion Injuries: Involvement of Anti-apoptotic and Antioxidant Activities.

Reperfusion after cerebral ischemia causes additional ischemic injuries due to sudden recovery of blood supply. It usually produces excessive reactive species, mitochondrial dysfunction, oxidative stress, and cell apoptosis. Our study is designed to examine the role of miR-421 antagomir in cerebral ischemia/reperfusion injuries, as well as its underlying mechanisms. Middle cerebral artery occlusion (MCAO) model was performed with male Sprague Dawley (SD) rats for the initiation of cerebral ischemia/reperfusion injuries. Malondialdehyde (oxidative stress marker) and superoxide dismutase (antioxidant enzyme) were measured as indicators for oxidative stress. Flow cytometry was utilized to evaluate the cell apoptosis effects from miR-421. miR-421 antagomir significantly decreased neurological deficits and infarction volumes. It also downregulated malondialdehyde contents, upregulated superoxide dismutase activities, promoted the expressions of myeloid cells leukemia-1 and B cells lymphoma-2, and downregulated the expressions of Bax in the ischemic cortex. In addition, miR-421targeted MCL1 to exert its biological functions. Our study indicated the neuroprotection effects of miR-421 antagomir on cerebral I/R injuries, which involved the suppression of cell apoptosis and oxidative stress. MiR-421 might provide a new therapeutic direction for ischemia/reperfusion injuries.

PMPCB Silencing Sensitizes HCC Tumor Cells to Sorafenib Therapy.

Hepatocellular carcinoma (HCC) tumors invariably develop resistance to cytotoxic and targeted agents, resulting in failed treatment and tumor recurrence. Previous in vivo short hairpin RNA (shRNA) screening evidence revealed mitochondrial-processing peptidase (PMPC) as a leading gene contributing to tumor cell resistance against sorafenib, a multikinase inhibitor used to treat advanced HCC. Here, we investigated the contributory role of the ß subunit of PMPC (PMPCB) in sorafenib resistance. Silencing PMPCB increased HCC tumor cell susceptibility to sorafenib therapy, decreased liver tumor burden, and improved survival of tumor-bearing mice receiving sorafenib. Moreover, sorafenib + PMPCB shRNA combination therapy led to attenuated liver tumor burden and improved survival outcome for tumor-bearing mice, and it reduced colony formation in murine and human HCC cell lines in vitro. Additionally, PMPCB silencing enhanced PINK1-Parkin signaling and downregulated the anti-apoptotic protein MCL-1 in sorafenib-treated HCC cells, which is indicative of a healthier pro-apoptotic phenotype. Higher pre-treatment MCL-1 expression was associated with inferior survival outcomes in sorafenib-treated HCC patients. Elevated MCL-1 expression was present in sorafenib-resistant murine HCC cells, while MCL-1 knockdown sensitized these cells to sorafenib. In conclusion, our findings advocate combination regimens employing sorafenib with PMPCB knockdown or MCL-1 knockdown to circumvent sorafenib resistance in HCC patients.

Alkaloids from Piper nigrum Synergistically Enhanced the Effect of Paclitaxel against Paclitaxel-Resistant Cervical Cancer Cells through the Downregulation of Mcl-1.

In the current study, nine amide alkaloids, including two new dimeric amides and a new natural product, were identified from Piper nigrum. Among them, seven compounds sensitized paclitaxel-resistant cervical cancer cells HeLa/PTX to paclitaxel. Piperine was a major component obtained from Piper nigrum, and its sensitization mechanism was investigated. Combination treatment enhanced cell apoptosis, which was mediated by downregulation of phospho-Akt and Mcl-1. Piperine (50 µM) combined with paclitaxel (200 nM) downregulated Mcl-1 protein expression with a decrease of 35.9 ± 9.5% ( P < 0.05). Moreover, overexpression of Mcl-1 attenuated the inhibitory effect of this combination. Furthermore, combination treatments of six dimeric amide alkaloids and paclitaxel all downregulated Mcl-1 protein expression with a decrease ranging from 23.5 ± 9.7% to 41.7 ± 7.2% ( P < 0.05). We reveal, for the first time, that dimeric amide alkaloids from plants possess a remarkable sensitization effect on cancer cells to paclitaxel.

Actinidia arguta (Hardy Kiwi) Root Extract Exerts Anti-cancer Effects via Mcl-1-Mediated Apoptosis in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive and chemoresistant liver malignancy. Thus, identification of strategies to overcome insensitivity to apoptosis and growth inhibition is a growing focus of research in this malignancy. This study evaluated the potential anti-cancer effects of an ethanol extract from the Actinidia arguta (Hardy Kiwi) root (RAE) on CCA. Our data demonstrated that RAE decreased cell viability and induced apoptosis by activation of Caspase 3, Caspase 8, and Poly (ADP-ribose) polymerase (PARP) in two CCA cell lines. RAE induced a decrease in Mcl-1 in cultured CCA cells and in xenograft CCA tumors. Administration of RAE every other day led to significant growth inhibition in tumor burden xenograft CCA mice. Western blotting analysis of paired human CCA and normal adjacent tissues from the same patient revealed that CCA tissues exhibited significantly higher Mcl-1 expression than normal tissues. Taken together, our findings demonstrated the anti-cancer effects of RAE on CCA both in vitro and in vivo. These data suggest that RAE may be a promising anti-CCA agent and could be beneficial in the treatment of CCA through the targeting of Mcl-1.

Humanized Mcl-1 mice enable accurate preclinical evaluation of MCL-1 inhibitors destined for clinical use.

Myeloid cell leukemia-1 (MCL-1) is a prosurvival B-cell lymphoma 2 (BCL-2) family member required for the sustained growth of many cancers. Recently, a highly specific MCL-1 inhibitor, S63845, showing sixfold higher affinity to human compared with mouse MCL-1, has been described. To accurately test efficacy and tolerability of this BH3-mimetic (BH3-only protein mimetic) drug in preclinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse strain in which MCL-1 was replaced with its human homolog. huMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to the MCL-1 inhibitor S63845. Importantly, nontransformed cells and lymphomas from huMcl-1;Eµ-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;Eµ-Myc lymphoma cells were transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide led to long-term remission in ∼60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMcl-1 mouse model for testing MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.

Combination therapy with protein kinase inhibitor H89 and Tetrandrine elicits enhanced synergistic antitumor efficacy.

BACKGROUND: Tetrandrine, a bisbenzylisoquinoline alkaloid that was isolated from the medicinal plant Stephania tetrandrine S. Moore, was recently identified as a novel chemotherapy drug. Tetrandrine exhibited a potential antitumor effect on multiple types of cancer. Notably, an enhanced therapeutic efficacy was identified when tetrandrine was combined with a molecularly targeted agent. H89 is a potent inhibitor of protein kinase A and is an isoquinoline sulfonamide. METHODS: The effects of H89 combined with tetrandrine were investigated in vitro with respect to cell viability, apoptosis and autophagy, and synergy was assessed by calculation of the combination index. The mechanism was examined by western blot, flow cytometry and fluorescence microscopy. This combination was also evaluated in a mouse xenograft model; tumor growth and tumor lysates were analyzed, and a TUNEL assay was performed. RESULTS: Combined treatment with H89 and tetrandrine exerts a mostly synergistic anti-tumor effect on human cancer cells in vitro and in vivo while sparing normal cells. Mechanistically, the combined therapy significantly induced cancer cell apoptosis and autophagy, which were mediated by ROS regulated PKA and ERK signaling. Moreover, Mcl-1 and c-Myc were shown to play a critical role in H89/tetrandrine combined treatment. Mcl-1 ectopic expression significantly diminished H89/tetrandrine sensitivity and amplified c-Myc sensitized cancer cells in the combined treatment. CONCLUSION: Our findings demonstrate that the combination of tetrandrine and H89 exhibits an enhanced therapeutic effect and may become a promising therapeutic strategy for cancer patients. They also indicate a significant clinical application of tetrandrine in the treatment of human cancer. Moreover, the combination of H89/tetrandrine provides new selectively targeted therapeutic strategies for patients with c-Myc amplification.

Apoptosis induced by 9,11­dehydroergosterol peroxide from Ganoderma Lucidum mycelium in human malignant melanoma cells is Mcl­1 dependent.

9,11-Dehydroergosterol peroxide [9(11)-DHEP] is an important steroid from medicinal mushroom, which has been reported to exert antitumor activity in several tumor types. However, the role of 9(11)­DHEP toward the malignant melanoma cells has not been investigated. In the present study, the steroid from Ganoderma lucidum was purified on a submerged culture, and its antitumor mechanisms on A375 human malignant melanoma cells was investigated by MTT, flow cytometry and western blotting. The studies demonstrated that apoptotic mechanisms of the steroid were caspase­dependent and mediated via the mitochondrial pathway. The steroid did not cause significant changes in the expression levels of B­cell lymphoma 2 (Bcl­2) family proteins, Bcl­2­like protein 11, p53 upregulated modulator of apoptosis, Bcl­2­associated X protein, BH3 interacting­domain death agonist, Bcl­2­associated death promoter and Bcl­2, but it significantly downregulated induced myeloid leukemia cell differentiation protein Mcl­1 (Mcl­1) in melanoma cells, suggesting the key role of Mcl­1 in regulating apoptosis of melanoma cells induced by the steroid. These properties of 9(11)­DHEP advocate its usage as supplements in human malignant melanoma chemoprevention. The present study also suggests that mycelium of G. lucidum has a potential for producing bioactive substances and extracts with applications in medicine.

Molecular docking studies of bioactive compounds from Annona muricata Linn as potential inhibitors for Bcl-2, Bcl-w and Mcl-1 antiapoptotic proteins.

Annona muricata Linn or usually identified as soursop is a potential anticancer plant that has been widely reported to contain valuable chemopreventive agents known as annonaceous acetogenins. The antiproliferative and anticancer activities of this tropical and subtropical plant have been demonstrated in cell culture and animal studies. A. muricata L. exerts inhibition against numerous types of cancer cells, involving multiple mechanism of actions such as apoptosis, a programmed cell death that are mainly regulated by Bcl-2 family of proteins. Nonetheless, the binding mode and the molecular interactions of the plant's bioactive constituents have not yet been unveiled for most of these mechanisms. In the current study, we aim to elucidate the binding interaction of ten bioactive phytochemicals of A. muricata L. to three Bcl-2 family of antiapoptotic proteins viz. Bcl-2, Bcl-w and Mcl-1 using an in silico molecular docking analysis software, Autodock 4.2. The stability of the complex with highest affinity was evaluated using MD simulation. We compared the docking analysis of these substances with pre-clinical Bcl-2 inhibitor namely obatoclax. The study identified the potential chemopreventive agent among the bioactive compounds. We also characterized the important interacting residues of protein targets which involve in the binding interaction. Results displayed that anonaine, a benzylisoquinoline alkaloid, showed a high affinity towards the Bcl-2, thus indicating that this compound is a potent inhibitor of the Bcl-2 antiapoptotic family of proteins.

Arenobufagin Induces Apoptotic Cell Death in Human Non-Small-Cell Lung Cancer Cells via the Noxa-Related Pathway.

Arenobufagin, an active component isolated from the traditional Chinese medicine Chan Su, exhibits anticancer influences in several human malignancies. However, the effects and action mechanisms of arenobufagin on non-small-cell lung cancer (NSCLC) are still unknown. In this study, we reported that arenobufagin acted through activation of Noxa-related pathways and promoted apoptotic cell death in human NSCLC cells. Our results revealed that arenobufagin-induced apoptosis was caspase-dependent, as evidenced by the fact that caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) were cleaved, and pretreatment with a pan-caspase inhibitor Z-VAD-FMK inhibited the pro-apoptosis effect of arenobufagin. Mechanistically, we further found that arenobufagin rapidly upregulated the expression of the pro-apoptosis protein Noxa, and abrogated the anti-apoptosis protein Mcl-1, a major binding partner of Noxa in the cell. More importantly, the knockdown of Noxa greatly blocked arenobufagin-induced cell death, highlighting the contribution of this protein in the anti-NSCLC effects of arenobufagin. Interestingly, arenobufagin also increased the expression of p53, a direct transcriptional activator for the upregulation of the Noxa protein. Taken together, our results suggest that arenobufagin is a potential anti-NSCLC agent that triggers apoptotic cell death in NSCLC cells through interfering with the Noxa-related pathway.

NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin.

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis.

Small molecule inhibitor TW-37 is tolerable and synergistic with chemotherapy in nasopharyngeal carcinoma.

Chemotherapy is a crucial adjuvant therapy of advanced nasopharyngeal carcinoma (NPC). However, enhancing sensitivity and tolerance of chemotherapeutics in NPC treatment have been challenging. Both Bcl-2 and Mcl-1, 2 pro-survival proteins of Bcl-2 family, play essential roles on the chemotherapy tolerance of numerous cancers. In the present study, we explored the influences of TW-37, a small molecule inhibitor of Bcl-2 and Mcl-1, on the efficiency of chemotherapy for NPC. Oncomine cancer database shows that NPC tissues have higher expression of Bcl-2 and Mcl-1 than those of normal nasopharyngeal epithelial (NPE) tissues. And our results reveal that chemotherapeutics, Cisplatin (CDDP) and 5-Fluoracil (5-FU), result in the greater decrease of protein level of Bcl-2 and Mcl-1 in NPC cells than those in NPE cells. TW-37 does not have significant impact on the chemotherapeutics-treated NPE cell viability at a dosage that efficiently reduces chemotherapeutics-treated NPC cell viability. Moreover, impacts of TW-37 on the cell viability of chemotherapeutics-treated NPC cells are dependent on the expression of Bcl-2 and Mcl-1 in NPC cells. Further explorations suggest that TW-37 prominently promotes apoptosis in NPC cells under chemotherapeutics treatments but not in NPE cells. Meanwhile, TW-37 also remarkably reduces colony formation ability of chemotherapeutics-treated NPC cells. Importantly, in vivo models, TW-37 observably increases chemosensitivity of NPC tumors but has not markedly influence on the normal tissues in mice. In conclusion, our results point to TW-37 as a promising ancillary drug for the chemotherapy of NPC.

NRAS mutations in cutaneous T cell lymphoma (CTCL) sensitize tumors towards treatment with the multikinase inhibitor Sorafenib.

Therapy of cutaneous T cell lymphoma (CTCL) is complicated by a distinct resistance of the malignant T cells towards apoptosis that can be caused by NRAS mutations in late-stage patients. These mutations correlate with decreased overall survival, but sensitize the respective CTCL cells towards MEK-inhibition-induced apoptosis which represents a promising novel therapeutic target in CTCL. Here, we show that the multi-kinase inhibitor Sorafenib induces apoptosis in NRAS-mutated CTCL cells. CTCL cell lines and to a minor extent primary T cells from Sézary patients without NRAS mutations are also affected by Sorafenib-induced apoptosis suggesting a sensitizing role of NRAS mutations for Sorafenib-induced apoptosis. When combining Sorafenib with the established CTCL medication Vorinostat we detected an increase in cell death sensitivity in CTCL cells. The combination treatment acted synergistically in apoptosis induction in both non-mutant and mutant CTCL cells. Mechanistically, this synergistic apoptosis induction by Sorafenib and Vorinostat is based on the downregulation of the anti-apoptotic protein Mcl-1, but not of other Bcl-2 family members. Taken together, these findings suggest that Sorafenib in combination with Vorinostat represents a novel therapeutic approach for the treatment of CTCL patients.

FBW7 mutations mediate resistance of colorectal cancer to targeted therapies by blocking Mcl-1 degradation.

Colorectal cancer (CRC), the second leading cause of cancer-related deaths in the US, has been treated with targeted therapies. However, the mechanisms of differential responses and resistance of CRCs to targeted therapies are not well understood. In this study, we found that genetic alterations of FBW7, an E3 ubiquitin ligase and a tumor suppressor frequently mutated in CRCs, contribute to resistance to targeted therapies. CRC cells containing FBW7-inactivating mutations are insensitive to clinically used multi-kinase inhibitors of RAS/RAF/MEK/ERK signaling, including regorafenib and sorafenib. In contrast, sensitivity to these agents is not affected by oncogenic mutations in KRAS, BRAF, PIK3CA or p53. These cells are defective in apoptosis owing to blocked degradation of Mcl-1, a pro-survival Bcl-2 family protein. Deleting FBW7 in FBW7-wild-type CRC cells abolishes Mcl-1 degradation and recapitulates the in vitro and in vivo drug-resistance phenotypes of FBW7-mutant cells. CRC cells selected for regorafenib resistance have progressive enrichment of pre-existing FBW7 hotspot mutations, and are cross-resistant to other targeted drugs that induce Mcl-1 degradation. Furthermore, a selective Mcl-1 inhibitor restores regorafenib sensitivity in CRC cells with intrinsic or acquired resistance. Together, our results demonstrate FBW7 mutational status as a key genetic determinant of CRC response to targeted therapies, and Mcl-1 as an attractive therapeutic target.

Growth inhibitory and apoptosis-inducing effects of allergen-free Rhus verniciflua Stokes extract on A549 human lung cancer cells.

Evidence suggests that Rhus verniciflua Stokes (RVS) or its extract has the potential to be used for the treatment of inflammatory and neoplastic diseases. However, direct use of RVS or its extract as a herbal medicine has been limited due to the presence of urushiol, an allergenic toxin. In the present study, we prepared an extract of the allergen­removed RVS (aRVS) based on a traditional method and investigated its inhibitory effect on the growth of various types of human cancer cells, including lung (A549), breast (MCF-7) and prostate (DU-145) cancer cell lines. Notably, among the cell lines tested, treatment with the aRVS extract strongly inhibited proliferation of the A549 cells at a 0.5 mg/ml concentration for 24 h that was not cytotoxic to normal human dermal fibroblasts. Furthermore, aRVS extract treatment largely reduced the survival and induced apoptosis of the A549 cells. At the mechanistic levels, treatment with the aRVS extract led to the downregulation of Bcl-2 and Mcl-1 proteins, the activation of caspase-9/-3 proteins, an increase in cytosolic cytochrome c levels, the upregulation of Bax protein, an increase in phosphorylated p53 protein but a decrease in phosphorylated S6 protein in the A549 cells. Importantly, treatment with z-VAD­fmk, a pan-caspase inhibitor attenuated aRVS extract-induced apoptosis in the A549 cells. These results demonstrate firstly that aRVS extract has growth inhibitory and apoptosis-inducing effects on A549 human lung cancer cells through modulation of the expression levels and/or activities of caspases, Bcl-2, Mcl-1, Bax, p53 and S6.

Angelica polymorpha Maxim Induces Apoptosis of Human SH-SY5Y Neuroblastoma Cells by Regulating an Intrinsic Caspase Pathway.

Angelica polymorpha Maxim root extract (APRE) is a popular herbal medicine used for treating stomachache, abdominal pain, stomach ulcers, and rheumatism; however the effect of APRE on cancer cells has not yet been explored. Here, we examined APRE cytotoxicity seen on target neuroblastoma cells (NB) using cell viability assays, DAPI visualization of fragmented DNA, and Western blotting analysis of candidate signaling pathways involved in proliferation and apoptosis. We demonstrated that APRE reduced cell viability in NB to a greater extent than in fibroblast cells. In addition, we found that APRE could inhibit the three classes of MAPK proteins and could also down-regulate the PI3K/AKT/GSK-3ß activity all being relevant for proliferation and survival. APRE could also up-regulate Bax expression and down-regulate Bcl-2 and Mcl-1. With APRE treatment, depolarization of mitochondria membrane potential and activation of caspase-3 was demonstrated in the SH-SY5Y cells. We could not found increased activity of death receptor and caspase-8 as markers of the extrinsic apoptosis pathway for the APRE treated cells. In presence of a caspase-3 siRNA and a pan-caspase inhibitor, APRE could not reduce the viability of NB cells to a significant degree. So we predicted that with APRE, the intrinsic pathway was solely responsible for inducing apoptosis as we also showed that the non-caspase autophagy pathway or ER stress-ROS mediated pathways were not involved. These findings demonstrate that an intrinsic mitochondria-mediated apoptosis pathway mediates the apoptotic effects of APRE on SH-SY5Y cells, and that APRE shows promise as a novel agent for neuroblastoma therapy.

Momordica cochinchinensis Spreng. seed extract suppresses breast cancer growth by inducing cell cycle arrest and apoptosis.

The herb Momordica cochinchinensis has been used for a variety of purposes, and been shown to have anti­cancer properties. The present study assessed the potency and the underlying mechanisms of action of the ethyl acetate extract of seeds of Momordica cochinchinensis (ESMC2) on breast cancer cells. Therefore, the effects of ESMC2 on the cell viability, cell cycle and apoptosis of MDA­MB­231 cells were investigated. The results showed that ESMC2 exerted a marked growth inhibitory effect on the cells. Cell cycle arrest in G2 phase following treatment with ESMC2 was associated with a marked increase in the protein levels of cyclin B1, cyclin E and cyclin-dependent kinase 1 and a decrease in cyclin D1 expression. In addition, ESMC2 dose­dependently induced cell apoptosis, which was mediated via upregulation of the apoptosis-associated proteins p53, B-cell lymphoma 2 (Bcl­2)­associated X protein, Bcl-2 homologous antagonist killer and Bcl-2-associated death promoter expression, as well as downregulation of nuclear factor kappa B, Bcl­2 and myeloid cell leukemia­1. Furthermore, the activation of extracellular signal-regulated kinase 1/2, p38, c-Jun N-terminal kinase (JNK) and Akt phosphorylation were decreased by ESMC2 in a dose­dependent manner, indicating that ESMC2 exerted its effects via the mitogen-activated protein kinase/JNK pathway. Furthermore, nude mouse xenotransplant models were used to evaluate the tumor growth inhibitory effects of ESMC2. The possible chemical components of ESMC2 were analyzed by gas chromatography-mass spectrometry, and 12 compounds were detected from the major peaks based on the similarity index with entries of a compound database. The results of the present study may aid in the development of novel therapies for breast cancer.

Oncostatin M-dependent Mcl-1 induction mediated by JAK1/2-STAT1/3 and CREB contributes to bioenergetic improvements and protective effects against mitochondrial dysfunction in cortical neurons.

Oncostatin M (OSM), a cytokine in the interleukin-6 (IL-6) family, has been proposed to play a protective role in the central nervous system, such as attenuation of excitotoxicity induced by N-methyl-D-aspartate (NMDA) and glutamate. However, the potential neuroprotective effects of OSM against mitochondrial dysfunction have never been reported. In the present study, we tested the hypothesis that OSM may confer neuronal resistance against 3-nitropropionic acid (3-NP), a plant toxin that irreversibly inhibits the complex II of the mitochondrial electron transport chain, and characterized the underlying molecular mechanisms. We found that OSM preconditioning dose- and time-dependently protected cortical neurons against 3-NP toxicity. OSM stimulated expression of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 family member expressed in differentiating myeloid cells, that required prior phosphorylation of Janus kinase-1 (JAK1), JAK2, extracellular signal-regulated kinase-1/2 (ERK1/2), signal transducer and activator of transcription-3 (STAT3), STAT1, and cAMP-response element-binding protein (CREB). Pharmacological inhibitors of JAK1, JAK2, ERK1/2, STAT3, STAT1, and CREB as well as the siRNA targeting at STAT3 and Mcl-1 all abolished OSM-dependent 3-NP resistance. Finally, OSM-dependent Mcl-1 induction contributed to the enhancements of mitochondrial bioenergetics including increases in spare respiratory capacity and ATP production. In conclusion, our findings indicated that OSM induces Mcl-1 expression via activation of ERK1/2, JAK1/2, STAT1/3, and CREB; furthermore, OSM-mediated Mcl-1 induction contributes to bioenergetic improvements and neuroprotective effects against 3-NP toxicity in cortical neurons. OSM may thus serve as a novel neuroprotective agent against mitochondrial dysfunction commonly associated with pathogenic mechanisms underlying neurodegeneration.

Apoptosis induced by Ginkgo biloba (EGb761) in melanoma cells is Mcl-1-dependent.

Melanoma is an aggressive skin cancer. Unfortunately, there is currently no chemotherapeutic agent available to significantly prolong the survival of the most patients with metastatic melanomas. Here we report that the Ginkgo biloba extract (EGb761), one of the most widely sold herbal supplements in the world, potently induces apoptosis in human melanoma cells by disturbing the balance between pro- and anti-apoptosis Bcl-2 family proteins. Treatment with EGb761 induced varying degrees of apoptosis in melanoma cell lines but not in melanocytes. Induction of apoptosis was caspase-dependent and appeared to be mediated by the mitochondrial pathway, in that it was associated with reduction in mitochondrial membrane potential and activation of Bax and Bak. Although EGb761 did not cause significant change in the expression levels of the BH3-only Bcl-2 family proteins Bim, Puma, Noxa, and Bad, it significantly downregulated Mcl-1 in sensitive but not resistant melanoma cells, suggesting a major role of Mcl-1 in regulating apoptosis of melanoma cells induced by EGb761. Indeed, siRNA knockdown of Mcl-1 enhanced EGb761-induced apoptosis, which was associated with increased activation of Bax and Bak. Taken together, these results demonstrate that EGb761 kills melanoma cells through the mitochondrial apoptotic pathway, and that Mcl-1 is a major regulator of sensitivity of melanoma cells to apoptosis induced by EGb761. Therefore, EGb761 with or without in combination with targeting Mcl-1 may be a useful strategy in the treatment of melanoma.

Anticancer siRNA cocktails as a novel tool to treat cancer cells. Part (B). Efficiency of pharmacological action.

This paper examines a perspective to use newly engineered nanomaterials as effective and safe carriers for gene therapy of cancer. Three different groups of cationic dendrimers (PAMAM, phosphorus, and carbosilane) were complexed with anticancer siRNA and the biophysical properties of the dendriplexes created were analyzed. The potential of the dendrimers as nanocarriers for anticancer Bcl-xl, Bcl-2, Mcl-1 siRNAs and additionally a scrambled sequence siRNA has been explored. Dendrimer/siRNA complexes were characterised by various methods including fluorescence, zeta potential, dynamic light scattering, circular dichroism, gel electrophoresis and transmission electron microscopy. In this part of study, the transfection of complexes in HeLa and HL-60 cells was analyzed using both single apoptotic siRNAs and a mixture (cocktail) of them. Cocktails were more effective than single siRNAs, allowing one to decrease siRNAs concentration in treating cells. The dendrimers were compared as siRNA carriers, the most effective being the phosphorus-based ones. However, they were also the most cytotoxic on their own, so that in this regard the application of all dendrimers in anticancer therapy will be discussed.