Qing-Bing Zha

Cucurbitacin B induces rapid depletion of the G-actin pool through reactive oxygen species-dependent actin aggregation in melanoma cells

Cucurbitacin B (CuB), a triterpenoid compound isolated from Cucurbitaceae plants, has been reported as a promising anti-cancer agent, yet its action mechanism is still controversial. In this study, we explored the potential mechanism of CuB in murine B16F10 melanoma cells. Anti-proliferation and anti-invasion effects were assessed in cultured cells, and in vivo anti-tumor activity was evaluated in a murine subcutaneous melanoma model. Flow cytometry was adopted to analyze cell cycle distribution and reactive oxygen species (ROS) levels. Actin levels were determined by western blot analysis, and the profiles of differential expressed proteins were identified by a quantitative proteomic approach. The results showed that CuB exerted inhibitory effects on cell proliferation, colony formation, as well as migration and invasion potential of the melanoma cells. The growth of subcutaneous melanoma was significantly inhibited in mice treated with CuB when compared with control group. Furthermore, CuB treatment caused rapid cell membrane blebbing and deformation, and induced G(2)/M-phase arrest and formation of multiploid cells. Notably, the G-actin pool was rapidly depleted and actin aggregates were formed quickly after CuB treatment. A number of cytoskeleton-regulatory proteins were differentially regulated. Blockage of ROS production significantly reduced the G-actin depletion ability and the anti-tumor activity of CuB. These findings indicate that CuB induces rapid depletion of the G-actin pool through ROS-dependent actin aggregation in melanoma cells, which may at least partly account for its anti-tumor activity.

Histone deacetylase inhibitor valproic acid sensitizes B16F10 melanoma cells to cucurbitacin B treatment

Cucurbitacin B (CuB) is reported to have anti-proliferation effects on a variety of tumors including melanoma, and more effective regimens by combination of this agent with others are under investigation. In this study, the anti-melanoma effect of CuB as a single agent and in combination with valproic acid (VPA), an inhibitor of histone deacetylase (HDAC), was evaluated in B16F10, a mouse melanoma cell line. The results demonstrated that CuB inhibited the proliferation of the cell line in a dose-dependent manner. However, it was likely that a pro-survival compensatory response, involving the induction of autophagy and upregulation of anti-apoptotic Bcl-2 protein, was induced by CuB treatment, which might greatly decrease the cytotoxicity of this agent. Supporting this, the melanoma cells were found to be more sensitive to the combination of CuB with chloroquine, a well-known autophagy inhibitor. And CuB-induced autophagy was associated with c-Jun N-terminal kinase (JNK) activation, at least partly, since inhibition of JNK activity by SP600125 could alleviate the autophagy. When CuB was combined with VPA, the two drugs showed synergistic cytotoxicity by induction of cell apoptosis. Moreover, the multiploidization effect of CuB was also suppressed in the presence of VPA. In contrast to the transient activation of JNKs by CuB, the combination of CuB and VPA resulted in prolonged JNK activation, although at low level after 4 h. Our results demonstrated that HDAC inhibitor VPA can sensitize B16F10 cells to CuB treatment through induction of apoptotic pathway.

Valproic acid synergistically enhances the cytotoxicity of gossypol in DU145 prostate cancer cells: an iTRTAQ-based quantitative proteomic analysis.

Gossypol (GOS), a BH3 mimetic, has been investigated as a sensitizing co-therapy to radiation and chemotherapy in treatment of metastatic prostate cancer. In this study, we found that valproic acid (VPA), a histone deacetylase inhibitor (HDACI), counteracted the suppressive effect of GOS on histone H3 acetylation and enhanced the cytotoxicity of GOS to DU145 prostate cancer cells. Significant synergistic effects were observed in combined GOS and VPA treatment, culminating in more DNA damage and cell death. The iTRAQ-based quantitative proteomic analysis revealed differential proteomic profiles in cells treated with VPA, GOS or their combination. In GOS-treated cells, oxidative phosphorylation-related proteins were depressed and endoplasmic reticulum stress markers were upregulated. In the presence of VPA, the GOS-induced mitochondrial stress was further enhanced since glycolysis- and hypoxia-associated proteins were upregulated, suggesting a disruption of energy metabolism in these cells. Furthermore, the DNA damage repair ability of cells co-treated with GOS and VPA was also decreased, as evidenced by the downregulation of DNA damage repair proteins and the enhancement of DNA fragmentation and cell death. These findings suggest that GOS in combination with an HDACI has the potential to increase its clinical efficacy in the treatment of prostate cancer.

Formation of cofilin-actin rods following cucurbitacin-B-induced actin aggregation depends on Slingshot homolog 1-mediated cofilin hyperactivation.

Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e., activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod‐like aggregates, which were highly colocalized with actin rods, indicating the formation of cofilin–actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase Slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB‐induced cofilin hyperactivation and cofilin–actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol‐containing reactive oxygen species (ROS) scavenger N‐acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB‐induced actin aggregation, cofilin hyperactivation and cofilin–actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB‐induced formation of cofilin–actin rods was mediated by SSH1‐dependent but CIN‐independent cofilin hyperactivation. J. Cell. Biochem. 114: 2415–2429, 2013.

ATP-Induced Inflammasome Activation and Pyroptosis Is Regulated by AMP-Activated Protein Kinase in Macrophages

Adenosine triphosphate (ATP) is released by bacteria and host cells during bacterial infection as well as sterile tissue injury, acting as an inducer of inflammasome activation. Previous studies have shown that ATP treatment leads to AMP-activated protein kinase (AMPK) activation. However, it is unclear whether AMPK signaling has been involved in the regulation of ATP-induced inflammasome activation and subsequent pyroptosis. In this study, we aimed to investigate this issue in lipopolysaccharide-activated murine macrophages. Our results showed that AMPK signaling was activated in murine macrophages upon ATP treatment, which was accompanied by inflammasome activation and pyroptosis as evidenced by rapid cell membrane rupture as well as mature interleukin (IL)-1β and active caspase-1p10 release. The ATP-induced inflammasome activation and pyroptosis were markedly suppressed by an AMPK inhibitor compound C or small-interfering RNA-mediated knockdown of AMPKα, but could be greatly enhanced by metformin (a well-known AMPK agonist). Importantly, metformin administration increased the mortality of mice with bacterial sepsis, which was likely because metformin treatment enhanced the systemic inflammasome activation as indicated by elevated serum and hepatic IL-1β levels. Collectively, these data indicated that the AMPK signaling positively regulated ATP-induced inflammasome activation and pyroptosis in macrophages, highlighting the possibility of AMPK-targeting therapies for inflammatory diseases involving inflammasome activation.

Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity

Cucurbitacins, the natural triterpenoids possessing many biological activities, have been reported to suppress the mTORC1/p70S6K pathway and to induce autophagy. However, the correlation between such activities is largely unknown. In this study, we addressed this issue in human cancer cells in response to cucurbitacin E (CuE) treatment. Our results showed that CuE induced autophagy as evidenced by the formation of LC3-II and colocalization of punctate LC3 with the lysosomal marker LAMP2 in HeLa and MCF7 cells. However, CuE induced much lower levels of autophagy in ATG5-knocked down cells and failed to induce autophagy in DU145 cells lacking functional ATG5 expression, suggesting the dependence of CuE-induced autophagy on ATG5. Consistent with autophagy induction, mTORC1 activity (as reflected by p70S6K and ULK1S758 phosphorylation) was inhibited by CuE treatment. The suppression of mTORC1 activity was further confirmed by reduced recruitment of mTOR to the lysosome, which is the activation site of mTORC1. In contrast, CuE rapidly activated AMPK leading to increased phosphorylation of its substrates. AMPK activation contributed to CuE-induced suppression of mTORC1/p70S6K signaling and autophagy induction, since AMPK knockdown diminished these effects. Collectively, our data suggested that CuE induced autophagy in human cancer cells at least partly via downregulation of mTORC1 signaling and upregulation of AMPK activity.

High Frequencies Cytomegalovirus pp65495–503-Specific CD8+ T Cells in Healthy Young and Elderly Chinese Donors: Characterization of Their Phenotypes and TCR Vβ Usage

Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus which persists lifelong after primary infection and can lead to a significant disease in the immunocompromised individuals. CD8+ T cells are believed to play a crucial role in both the elimination of active infection and maintenance of HCMV latency. Large expansions of CD8+ T cells specific for a single epitope of HCMV have been well documented in Caucasoid population. To date, no similar study has been performed in Chinese populations. Here we report the characteristics of HCMV-specific CD8+ T cells in healthy young and elderly Chinese donors using pp65495–503-loaded HLA-A*0201 tetramers. Cells were stained with a combination of the tetramers and antibodies for CD28 and CD57 or a panel of TCR Vβ and analyzed by three-color flow cytometry. The frequencies of pp65495–503-specific T cells within total CD8+ T cell population were between 0.14 and 6.84% (mean 2.45%) in the young donors and were from 0.33 to 6.89% (mean 1.95%) in the elderly donors, respectively. There was no significant difference between the two groups. The expression of CD28 was decreased whereas CD57 expression was increased in tetramer-negative CD8+ T cells in the elderly when compared with the young group. However, neither of these changes was found within tetramer-positive cell populations. Moreover, TCR Vβ usage within tetramer-positive population was predominated by certain TCR Vβ subsets. These results demonstrate that large expansions of HCMV-specific CD8+ T cells with certain subsets TCR Vβ exist both in the healthy young and in the elderly Chinese individuals, which may play a role in the maintenance of virus latency but have potential detrimental influence on the immune responses to other pathogens or vaccinations.

Gossypol induces pyroptosis in mouse macrophages via a non-canonical inflammasome pathway.

Gossypol, a polyphenolic compound isolated from cottonseeds, has been reported to possess many pharmacological activities, but whether it can influence inflammasome activation remains unclear. In this study, we found that in mouse macrophages, gossypol induced cell death characterized by rapid membrane rupture and robust release of HMGB1 and pro-caspase-11 comparable to ATP treatment, suggesting an induction of pyroptotic cell death. Unlike ATP, gossypol induced much low levels of mature interleukin-1β (IL-1β) secretion from mouse peritoneal macrophages primed with LPS, although it caused pro-IL-1β release similar to that of ATP. Consistent with this, activated caspase-1 responsible for pro-IL-1β maturation was undetectable in gossypol-treated peritoneal macrophages. Besides, RAW 264.7 cells lacking ASC expression and caspase-1 activation also underwent pyroptotic cell death upon gossypol treatment. In further support of pyroptosis induction, both pan-caspase inhibitor and caspase-1 subfamily inhibitor, but not caspase-3 inhibitor, could sharply suppress gossypol-induced cell death. Other canonical pyroptotic inhibitors, including potassium chloride and N-acetyl-l-cysteine, could suppress ATP-induced pyroptosis but failed to inhibit or even enhanced gossypol-induced cell death, whereas nonspecific pore-formation inhibitor glycine could attenuate this process, suggesting involvement of a non-canonical pathway. Of note, gossypol treatment eliminated thioglycollate-induced macrophages in the peritoneal cavity with recruitment of other leukocytes. Moreover, gossypol administration markedly decreased the survival of mice in a bacterial sepsis model. Collectively, these results suggested that gossypol induced pyroptosis in mouse macrophages via a non-canonical inflammasome pathway, which raises a concern for its in vivo cytotoxicity to macrophages.

Piperine metabolically regulates peritoneal resident macrophages to potentiate their functions against bacterial infection

Pepper, a daily-used seasoning for promoting appetite, is widely used in folk medicine for treating gastrointestinal diseases. Piperine is the major alkaloid in pepper and possesses a wide range of pharmacological activities. However, the mechanism for linking metabolic and medicinal activities of piperine remains unknown. Here we report that piperine robustly boosts mTORC1 activity by recruiting more system L1 amino acid transporter (SLC7A5/SLC3A2) to the cell membrane, thus promoting amino acid metabolism. Piperine-induced increase of mTORC1 activity in resident peritoneal macrophages (pMΦs) is correlated with enhanced production of IL-6 and TNF-α upon LPS stimulation. Such an enhancement of cytokine production could be abrogated by inhibitors of the mTOR signaling pathway, indicating mTORs action in this process. Moreover, piperine treatment protected resident pMΦs from bacterium-induced apoptosis and disappearance, and increased their bacterial phagocytic ability. Consequently, piperine administration conferred mice resistance against bacterial infection and even sepsis. Our data highlight that piperine has the capacity to metabolically reprogram peritoneal resident macrophages to fortify their innate functions against bacterial infection.

Piperine Suppresses the Expression of CXCL8 in Lipopolysaccharide-Activated SW480 and HT-29 Cells via Downregulating the Mitogen-Activated Protein Kinase Pathways

The anti-inflammatory effect of piperine has been largely investigated in macrophages, but its activity on epithelial cells in inflammatory settings is unclear. The present study aimed to investigate the effect of piperine on the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated human epithelial-like SW480 and HT-29 cells. Our data showed that although piperine inhibited the proliferation of SW480 and HT-29 cells in a dose-dependent manner, it had low cytotoxicity on these cell lines with 50 % inhibiting concentration (IC50) values greater than 100 μM. As epithelial-like cells, SW480 and HT-29 cells secreted high levels of the chemokine CXCL8 upon LPS stimulation. Importantly, piperine dose-dependently suppressed LPS-induced secretion of CXCL8 and the expression of CXCL8 messenger RNA (mRNA). Although piperine failed to affect the critical inflammatory nuclear factor-κB pathway, it attenuated the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling. Consistent with previous reports, p38 signaling seemed to play a more pronounced role on the CXCL8 expression than JNK signaling since inhibition of p38, instead of JNK, greatly suppressed LPS-induced CXCL8 expression. Collectively, our results indicated that piperine could attenuate the inflammatory response in epithelial cells via downregulating the MAPK signaling and thus the expression of CXCL8, suggesting its potential application in anti-inflammation therapy.