Chia-Che Chang

Development of natural anti-tumor drugs by microorganisms

Discoveries of tumor-resistant pharmacological drugs have mainly resulted from screening of natural products and their analogs. Some are also discovered incidentally when studying organisms. The great biodiversity of microorganisms raises the possibility of producing secondary metabolites (e.g., mevastatin, lovastatin, epothilone, salinosporamide A) to cope with adverse environments. Recently, natural plant pigments with anti-tumor activities such as β-carotene, lycopene, curcumin and anthocyanins have been proposed. However, many plants have a long life cycle. Therefore, pigments from microorganisms represent another option for the development of novel anti-tumor drugs. Prodigiosin (PG) is a natural red pigment produced by microorganisms, i.e., Serratia marcescens and other gram-negative bacteria. The anti-tumor potential of PG has been widely demonstrated. The families of PG (PGs), which share a common pyrrolylpyrromethene (PPM) skeleton, are produced by various bacteria. PGs are bioactive pigments and are known to exert immunosuppressive properties, in vitro apoptotic effects, and in vivo anti-tumor activities. Currently the most common strain used for producing PGs is S. marcescens. However, few reports have discussed PGs production. This review therefore describes the development of an anti-tumor drug, PG, that can be naturally produced by microorganisms, and evaluates the microbial production system, fermentation strategies, purification and identification processes. The application potential of PGs is also discussed.

Imiquimod simultaneously induces autophagy and apoptosis in human basal cell carcinoma cells

Background  Imiquimod shows antitumour activity through the stimulation of cell‐mediated immunity in vivo. Recent studies have shown that imiquimod promotes apoptosis in melanoma cells and induces autophagy in macrophage cell lines.

Prodigiosin down-regulates survivin to facilitate paclitaxel sensitization in human breast carcinoma cell lines

Prodigiosin is a bacterial metabolite with potent anticancer activity, which is attributed to its proapoptotic effect selectively active in malignant cells. Still, the molecular mechanisms whereby prodigiosin induces apoptosis remain largely unknown. In particular, the role of survivin, a vital inhibitor of apoptosis, in prodigiosin-induced apoptosis has never been addressed before and hence was the primary goal of this study. Our results showed that prodigiosin dose-dependently induced down-regulation of survivin in multiple breast carcinoma cell lines, including MCF-7, T-47D and MDA-MB-231. This down-regulation is mainly regulated at the level of transcription, as prodigiosin reduced the levels of both survivin mRNA and survivin promoter activity but failed to rescue survivin expression when proteasome-mediated degradation is abolished. Importantly, overexpression of survivin rendered cells more resistant to prodigiosin, indicating an essential role of survivin down-regulation in prodigiosin-induced apoptosis. In addition, we found that prodigiosin synergistically enhanced cell death induced by paclitaxel, a chemotherapy drug known to up-regulate survivin that in turn confers its own resistance. This paclitaxel sensitization effect of prodigiosin is ascribed to the lowering of survivin expression, because prodigiosin was shown to counteract survivin induction by paclitaxel and, notably, the sensitization effect was severely abrogated in cells that overexpress survivin. Taken together, our results argue that down-regulation of survivin is an integral component mediating prodigiosin-induced apoptosis in human breast cancer cells, and further suggest the potential of prodigiosin to sensitize anticancer drugs, including paclitaxel, in the treatment of breast cancer.

Prodigiosin down-regulates SKP2 to induce p27KIP1 stabilization and antiproliferation in human lung adenocarcinoma cells

BACKGROUND AND PURPOSE High levels of SKP2 are a poor prognostic factor in multiple human cancers and mostly correlate with low p27KIP1 levels. Prodigiosin is a bacterial tripyrrole pigment with strong pro‐apoptotic activity. Induction of cell cycle blockade underlies one of its anticancer actions but the mechanisms involved are unclear. The aim of this study was to explore the role of the SKP2–p27KIP1 axis in prodigiosins cytostatic effect on human lung adenocarcinoma cells.

Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5 through the ROS-JNK-CHOP Signaling Axis in Human Ovarian Carcinoma Cell Lines.

Tanshinone IIA (TIIA) extracted from Salvia miltiorrhiza has been shown to possess antitumor and TRAIL-sensitizing activity. The involvement of DR5 in the mechanism whereby TIIA exerts its effects is unknown. This study aimed to explore the mechanism underlying TIIA augmentation of TRAIL-induced cell death in ovarian carcinoma cells. Cell viability was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of relating signaling proteins. Transcriptional activation was explored by a dual-luciferase reporter assay. We found that TIIA sensitized human ovarian carcinoma cells to TRAIL-induced extrinsic apoptosis. Combined treatment with subtoxic concentrations of TIIA and TRAIL was more effective than single treatments with respect to cytotoxicity, clonogenic inhibition, and the induction of caspase-8 and PARP activity in ovarian carcinoma cell lines TOV-21G and SKOV3. TIIA induced DR5 protein and mRNA expression in a concentration-dependent manner. DR5/Fc treatment markedly suppressed the TRAIL cytotoxicity enhanced by TIIA. These results indicate that DR5 plays an essential role in TIIA-induced TRAIL sensitization and that induction of DR5 by TIIA is mediated through the up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP gene expression by shRNA attenuated DR5 up-regulation and rescued cell viability under the treatment of TIIA-TRAIL combination. TIIA promoted JNK-mediated signaling to up-regulated CHOP and thereby inducing DR5 expression as shown by the ability of a JNK inhibitor to potently suppress the TIIA-mediated activation of CHOP and DR5. In addition, the quenching of ROS using NAC prevented the induction of JNK phosphorylation and CHOP induction. Furthermore, inhibition of ROS by NAC significantly attenuated TRAIL sensitization by TIIA. Taken together, these data suggest that TIIA enhances TRAIL-induced apoptosis by upregulating DR5 receptors through the ROS-JNK-CHOP signaling axis in human ovarian carcinoma cells.

Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosins capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2α phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosins tumoricidal effect. Mechanistically, prodigiosin engages the IRE1-JNK and PERK-eIF2α branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death.

Krüppel-like factor 4 is involved in cell scattering induced by hepatocyte growth factor

Summary Hepatocyte growth factor/scatter factor (HGF) is unique by inducing epithelial cell scattering, a cellular event pivotal to HGF-mediated invasive-growth response essential for embryonic development and metastasis. Krüppel-like factor 4 (KLF4) is a multifunctional zinc-finger transcription factor involved in cell proliferation, differentiation and self-renewal. We herein present the first evidence for the functional connection between KLF4 and HGF-induced cell scattering. In particular, we found that KLF4 was upregulated by HGF in two independent epithelial cell types, HepG2 and MDCK, whereas KLF4 knockdown inhibited HGF-induced E-cadherin suppression and cell scattering. Moreover, enforced nuclear KLF4 expression alone was sufficient to upregulate KLF4, downregulate E-cadherin and trigger scattering. Chromatin immunoprecipitation (ChIP) analysis further revealed that KLF4 induced suppression of E-cadherin transcription by directly binding to the E-cadherin promoter. Additionally, we proved that HGF-induced upregulation of KLF4 transcription and cell scattering require activation of the MEK/ERK signaling pathway and the induction of early growth response 1 (EGR-1). At the mechanistic level, ChIP analysis validated a direct binding of EGR-1 to the KLF4 promoter to induce KLF4 transcription; in turn, EGR-1-induced KLF4 binds to its own promoter, thus creating a positive feedback mechanism to sustain KLF4 expression and the resultant cell scattering. We conclude that KLF4 upregulation by HGF represents a novel mechanism mediating HGF-induced cell scattering and perhaps other associated events such as cell migration and invasion.

Prodigiosin inhibits gp91(phox) and iNOS expression to protect mice against the oxidative/nitrosative brain injury induced by hypoxia-ischemia.

This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100μg/kg, i.v.) at 1h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91(phox)), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood-brain barrier (BBB) by activation of nuclear factor-kappa B (NF-κB). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91(phox) and iNOS via activation of the NF-κB pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91(phox) and iNOS expression possibly by impairing NF-κB activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosins protective effect in the MCAo/r mice.

p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells.

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cells p53 status.

A promising "TRAIL" of tanshinones for cancer therapy.

An ideal cancer therapy specifically targets cancer cells while sparing normal tissues. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) elicits apoptosis by engaging its cognate death receptors (DRs—namely, DR4 and DR5. The cancer cell-selective proapoptotic action of TRAIL is highly attractive for cancer therapy, but clinical application of TRAIL is rather limited due to tumors’ inherent or acquired TRAIL resistance. Combining TRAIL with agents that reverse resistance to it has proved promising in the sensitization of TRAIL-induced apoptosis. Noteworthy, natural compounds have already been validated as potential resources for TRAIL sensitizers. In this review, we focus on the recently identified TRAILsensitizing effect of tanshinones, the anticancer ingredients of the medicinal plant Salvia miltiorrhiza (Danshen in Chinese). Research from our laboratories and others have revealed the synergy of a tanshinones-TRAIL combination in diverse types of cancer cells through up-regulation of DR5 and/or down-regulation of antiapoptotic proteins such as survivin. Thus, in addition to their anticancer mechanisms, tanshinones as TRAIL sensitizers hold great potential to be translated to TRAIL-based therapeutic modalities for combatting cancer.