BMX, a specific HDAC8 inhibitor, with TMZ for advanced CRC therapy: a novel synergic effect to elicit p53-, ß-catenin- and MGMT-dependent apoptotic cell death.

BACKGROUND: Despite advances in treatment, patients with refractory colorectal cancer (CRC) still have poor long-term survival, so there is a need for more effective therapeutic options. METHODS: To evaluate the HDAC8 inhibition efficacy as a CRC treatment, we examined the effects of various HDAC8 inhibitors (HDAC8i), including BMX (NBM-T-L-BMX-OS01) in combination with temozolomide (TMZ) or other standard CRC drugs on p53 mutated HT29 cells, as well as wild-type p53 HCT116 and RKO cells. RESULTS: We showed that HDAC8i with TMZ cotreatment resulted in HT29 arrest in the S and G2/M phase, whereas HCT116 and RKO arrest in the G0/G1 phase was accompanied by high sub-G1. Subsequently, this combination approach upregulated p53-mediated MGMT inhibition, leading to apoptosis. Furthermore, we observed the cotreatment also enabled triggering of cell senescence and decreased expression of stem cell biomarkers. Mechanistically, we found down-expression levels of ß-catenin, cyclin D1 and c-Myc via GSK3ß/ß-catenin signaling. Intriguingly, autophagy also contributes to cell death under the opposite status of ß-catenin/p62 axis, suggesting that there exists a negative feedback regulation between Wnt/ß-catenin and autophagy. Consistently, the Gene Set Enrichment Analysis (GSEA) indicated both apoptotic and autophagy biomarkers in HT29 and RKO were upregulated after treating with BMX. CONCLUSIONS: BMX may act as a HDAC8 eraser and in combination with reframed-TMZ generates a remarkable synergic effect, providing a novel therapeutic target for various CRCs. Video Abstract.

Design, synthesis and antitumour evaluation of novel anthraquinone derivatives.

We report the design, synthesis, and biological evaluation of 13 new and 1 known anthraquinone derivatives which exerted cytotoxicity against PC3, A549 and NTUB1 cell lines. The results indicate that, among these 14, compounds-1 and 14 showed the highest growth inhibitory effect on NTUB1 and PC3 cells, respectively. Compound-1 at lower doses targets DNA, induces DNA damage and subsequently triggers G2/M arrest and apoptotic cell death at 24 h. Previously we reported that 14 induced PC3 cell autophagy and in treated PC3 cells, cleaved caspase-3 and cleaved PARP, and survivin did not increase and increase, respectively. The autophagic and necrotic cell deaths mediated by 14-triggered ROS generation. Our study is the first to investigate the biological mechanism of 14 action in detail. We find that when 14 was co-administrated with Bafilomycin A1 (BAF) in PC3 cells, rapid necrotic cell death occurred with no cleaved caspase-3 and cleaved PARP activation and increasing the expression of survivin. We further show that necrotic signaling in these cells coincided with production of reactive oxygen species. In the present study, we developed methods to synthesize five new 14 analogues for studing the structure-activity relationships. This study could provide valuable sight to find new antitumor agents for cancer therapy.

Hepatocellular carcinoma-related cyclin D1 is selectively regulated by autophagy degradation system.

Dysfunction of degradation machineries causes cancers, including hepatocellular carcinoma (HCC). Overexpression of cyclin D1 in HCC has been reported. We previously reported that autophagy preferentially recruits and degrades the oncogenic microRNA (miR)-224 to prevent HCC. Therefore, in the present study, we attempted to clarify whether cyclin D1 is another oncogenic factor selectively regulated by autophagy in HCC tumorigenesis. Initially, we found an inverse correlation between low autophagic activity and high cyclin D1 expression in tumors of 147 HCC patients and three murine models, and these results taken together revealed a correlation with poor overall survival of HCC patients, indicating the importance of these two events in HCC development. We found that increased autophagic activity leads to cyclin D1 ubiquitination and selective recruitment to the autophagosome (AP) mediated by a specific receptor, sequestosome 1 (SQSTM1), followed by fusion with lysosome and degradation. Autophagy-selective degradation of ubiquitinated cyclin D1 through SQSTM1 was confirmed using cyclin D1/ubiquitin binding site (K33-238 R) and phosphorylation site (T286A) mutants, lentivirus-mediated silencing autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and Sqstm1 knockout cells. Functional studies revealed that autophagy-selective degradation of cyclin D1 plays suppressive roles in cell proliferation, colony, and liver tumor formation. Notably, an increase of autophagic activity by pharmacological inducers (amiodarone and rapamycin) significantly suppressed tumor growth in both the orthotopic liver tumor and subcutaneous tumor xenograft models. Our findings provide evidence of the underlying mechanism involved in the regulation of cyclin D1 by selective autophagy to prevent tumor formation. CONCLUSION: Taken together, our data demonstrate that autophagic degradation machinery and the cell-cycle regulator, cyclin D1, are linked to HCC tumorigenesis. We believe these findings may be of value in the development of alternative therapeutics for HCC patients. (Hepatology 2018;68:141-154).

Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt/ß-Catenin Signaling Pathway in Glioma Cells.

Thioridazine (THD) is a common phenothiazine antipsychotic drug reported to suppress growth in several types of cancer cells. We previously showed that THD acts as an antiglioblastoma and anticancer stem-like cell agent. However, the signaling pathway underlying autophagy and apoptosis induction remains unclear. THD treatment significantly induced autophagy with upregulated AMPK activity and engendered cell death with increased sub-G1 in glioblastoma multiform (GBM) cell lines. Notably, through whole gene expression screening with THD treatment, frizzled (Fzd) proteins, a family of G-protein-coupled receptors, were found, suggesting the participation of Wnt/ß-catenin signaling. After THD treatment, Fzd-1 and GSK3ß-S9 phosphorylation (inactivated form) was reduced to promote ß-catenin degradation, which attenuated P62 inhibition. The autophagy marker LC3-II markedly increased when P62 was released from ß-catenin inhibition. Additionally, the P62-dependent caspase-8 activation that induced P53-independent apoptosis was confirmed by inhibiting T-cell factor/ß-catenin and autophagy flux. Moreover, treatment with THD combined with temozolomide (TMZ) engendered increased LC3-II expression and caspase-3 activity, indicating promising drug synergism. In conclusion, THD induces autophagy in GBM cells by not only upregulating AMPK activity, but also enhancing P62-mediated autophagy and apoptosis through Wnt/ß-catenin signaling. Therefore, THD is a potential alternative therapeutic agent for drug repositioning in GBM.

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT-29 cells. In the present study, we observed that many autophagy-related genes in GFC-treated HT-29 cells were up- and down-regulated using a cDNA microarray containing oncogenes and kinase genes. GFC-induced autophagy of HT-29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double-membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5-Atg12 and PI3K/Beclin-1 complexes were observed using Western blot. Administration of autophagy inhibitor (3-methyladenine and shRNA Atg5) and apoptosis inhibitor Z-VAD showed that the GFC-induced autophagy was cytotoxic form and GFC-induced apoptosis enhanced GFC-induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC-induced anticancer mechanisms of human colorectal cancer.

Biosorption of copper ions from aqueous solution using rape straw powders: Optimization, equilibrium and kinetic studies.

In this paper, the adsorption behaviors of Cu(II) from the aqueous solution using rape straw powders were studied. The effects of initial Cu(II) concentration, pH range and absorbent dosage on the adsorption efficiency of Cu(II) by rape straw powder were investigated by Box-Behnken Design based on response surface methodology. The values of coefficient constant of the nonlinear models were 0.9997, 0.9984 and 0.9944 for removal Cu(II) from aqueous solution using rape straw shell, seed pods and straw pith core, respectively, which could navigate the design space for various factors on effects of biosorption Cu(II) from aqueous solution. The various factors of pH and biosorbents dosage were the key factors that affecting the removal efficiency of Cu(II) from aqueous solution. The biosorption equilibrium data presented its favorable monolayer adsorption Cu(II) onto shell, seed pods and straw pith core, respectively. The pseudo-second order kinetic model was the proper approach to determine the adsorption kinetics. The biosorption of Cu(II) onto surfaces of rape straw powders were confirmed and ion-exchanged in the adsorption process by energy dispersive spectrometer. The critical groups, -OH, -CH, -NH3+, -CH3, -NH and -C-O, exhibited by the infrared spectra results, changed to suggest that these groups played critical roles, especially -CH3 in the adsorption of copper ions onto rape straw powders. The study provided evidences that rape straw powders can be used for removing Cu(II) from aqueous water.

A potent indolylquinoline alleviates growth of human lung cancer cell tumorspheres.

To fight cancer at its roots by targeting cancer stem cells is a promising approach for therapy. Previously, an indolylquinoline derivative, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), was reported effectively inhibiting the growth of lung cancer cells through impairment of cellular mitochondria functions. To address more on drug efficiency, the study further exploited if EMMQ can impede the propagation of tumorspheres stemmed from non-small cell lung cancer cells. EMMQ inhibited proliferation of spheroids in culture. In animal models, administration of the drug attenuated the spheroid tumorigenicity. The activated apoptosis alleviated growth of xenograft tumors in immune-deficient mice as established by the enriched tumorspheres. More evidence suggested that the reduced stemness of the spheroid tumors is attributed to apoptotic death. The findings supported that EMMQ is an eligible approach to eradicate the minor but tumorigenic lung cancer tumorspheres.

Justicidin A-induced autophagy flux enhances apoptosis of human colorectal cancer cells via class III PI3K and Atg5 pathway.

Our previous reports showed that justicidin A (JA), a novel and pure arylnaphthalide lignan isolated from Justicia procumbens, induces apoptosis of human colorectal cancer cells and hepatocellular carcinoma cells, leading to the suppression of both tumor cell growth in NOD-SCID mice. Here, we reveal that JA induces autophagy in human colorectal cancer HT-29 cells by conversion of autophagic marker LC3-I to LC3-II. Furthermore, LC3 puncta and autophagic vesicle formation, and SQSTM1/p62 suppression were observed. Administration of autophagy inhibitor (bafilomycin A1 and chloroquine) and transfection of a tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) demonstrated that JA induces autophagy flux in HT-29 cells. Expression of LC3, SQSTM1, Beclin 1, and nuclear DNA double-strand breaks (representing apoptosis) were also detected in the tumor tissue of HT-29 cells transplanted into NOD-SCID mice orally administrated with JA. In addition, the expression of autophagy signaling pathway-related molecules p-PDK1, p-mTOR, p-p70S6k/p-RPS6KB2 was decreased, whereas that of class III PI3K, Beclin 1, Atg5-Atg12, and mitochondrial BNIP3 was increased in response to JA. Pre-treatment of the cells with class III PI3K inhibitor 3-methyladenine or Atg5 shRNA attenuated JA-induced LC3-II expression and LC3 puncta formation, indicating the involvement of class III PI3K and Atg5. A novel mechanism was demonstrated in the anticancer compound JA; pre-treatment with 3-methyladenine or Atg5 shRNA blocked JA-induced suppression in cell growth and colony formation, respectively, via inhibition of apoptosis. In contrast, administration of apoptosis inhibitor Z-VAD did not affect JA-induced autophagy. Our data suggest the chemotherapeutic potential of JA for treatment of human colorectal cancer.

An indolylquinoline derivative promotes apoptosis in human lung cancer cells by impairing mitochondrial functions.

A number of effective anti-cancer drugs contain either indole or quinoline group. Compounds fused indole and quinoline moieties altogether as indolylquinoline were rarely reported as anti-cancer agents. We reported here that a synthetic indolylquinoline derivative, 3-((7-ethyl-1H-indol-3-yl)-methyl)-2-methylquinoline (EMMQ), inhibited the growth of human non-small cell lung cancer (NSCLC) cells in dose- and time-dependent manners. The cytotoxicity was mediated through apoptotic cell death that began with mitochondrial membrane potential interruption and DNA damage. EMMQ caused transient elevation of p53 that assists in cytochrome c release, cleavage of downstream PARP and procaspase-3 and mitochondria-related apoptosis. The degree of apoptotic cell death depends on the status of tumor suppressor p53 of the target cells. H1299 cells with stable ectopic expression of p53 induced cytotoxicity by disrupting mitochondria functions that differed with those transfected with mutant p53. Knocking-down of p53 attenuated drug effects. EMMQ suppressed the growth of A549 tumor cells in xenograft tumors by exhibiting apoptosis characteristics. Given its small molecular weight acting as an effective p53 regulator in NSCLC cells, EMMQ could be an addition to the current list of lung cancer treatment.

Curcumin-enhanced chemosensitivity of FDA-approved platinum (II)-based anti-cancer drugs involves downregulation of nuclear endonuclease G and NF-κB as well as induction of apoptosis and G2/M arrest.

Curcumin, an active natural compound in turmeric and curry, has been reported to exhibit anti-cancer effect. Cisplatin, carboplatin and oxaliplatin are used to treat various types of cancers. However, acquired resistance and toxicities are observed. Here, the addition of curcumin significantly increased cytotoxicity of the anti-cancer drugs on human colorectal cancer HT-29 cells, producing synergistic (cisplatin and carboplatin) and additivity (oxaliplatin) effects. Treatments in combination with curcumin resulted in a significantly increased induction of apoptosis and occurrence of G2/M arrest. Nuclear apoptosis-inducing factor (AIF), EndoG and NF-κB were elevated by anti-cancer drugs, suggesting the involvement of AIF and EndoG. The addition of curcumin suppressed nuclear AIF and EndoG and reversed anti-cancer drugs-induced NF-κB expression, suggesting the association of EndoG and NF-κB in curcumin-enhanced chemosensitivity. Therefore, the intake of foods rich in curcumin or curcumin-containing supplements should be taken into consideration for patients receiving chemotherapy to optimize the outcome of treatments.

Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53.

In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells.

Formosanin C suppresses cancer cell proliferation and migration by impeding autophagy machinery.

Formosanin C (FC) is a natural compound extracted from Paris formosana Hayata with anticancer activity. FC induces both autophagy and apoptosis in human lung cancer cells. FC-induced depolarization of mitochondrial membrane potential (MMP) may trigger mitophagy. In this study, we clarified the effect of FC on autophagy, mitophagy, and the role of autophagy in FC-related cell death and motility. We found FC caused the continuous increase of LC3 II (representing autophagosomes) from 24 to 72 h without degradation after treatment of lung and colon cancer cells, indicating that FC blocks autophagic progression. In addition, we confirmed that FC also induces early stage autophagic activity. Altogether, FC is not only an inducer but also a blocker of autophagy progression. Moreover, FC increased MMP accompanied by overexpression of COX IV (mitochondria marker) and phosphorylated Parkin (p-Parkin, mitophagy marker) in lung cancer cells, but no colocalization of LC3 with COX IV or p-Parkin was detected under confocal microscopy. Moreover, FC could not block CCCP (mitophagy inducer)-induced mitophagy. These results imply that FC disrupts mitochondria dynamics in the treated cells, and the underlying mechanism deserves further exploration. Functional analysis reveals that FC suppresses cell proliferation and motility through apoptosis and EMT-related pathway, respectively. In conclusion, FC acts as an inducer as well as a blocker of autophagy that results in cancer cell apoptosis and decreased motility. Our findings shed the light on the development of combined therapy with FC and clinical anticancer drugs for cancer treatment.

Synthetic lethality in human bladder cancer cells by curcumin via concurrent Aurora A inhibition and autophagy induction.

Combination therapies to induce mixed-type cell death and synthetic lethality have the potential to overcome drug resistance in cancer. In this study, we demonstrated that the curcumin-enhanced cytotoxicity of cisplatin/carboplatin in combination with gemcitabine was associated with Aurora A suppression-mediated G2/M arrest, and thus apoptosis, as well as MEK/ERK-mediated autophagy in human bladder cancer cells. Animal study data confirmed that curcumin combined with cisplatin/gemcitabine reduced tumorigenesis of xenograft in mice and this phenomenon was associated with elevated expressions of p-ERK and reduced p-Aurora A in tumors. Gene analyses using data repositories further revealed that reduced Aurora A expression alone did not significantly elevate the sensitivity of human bladder carcinoma cells to these anticancer drugs. Unlike other major cancer types, human bladder urothelial carcinoma tissue coexpressed higher AURKA and lower MAP1LC3B than normal tissue, and reduced Aurora A and induction of autophagy have been clinically associated with a better prognosis in patients with early but not advanced stage bladder cancer. Therefore, our results suggest that treatment strategies can utilize the synthetic lethal pair to concurrently suppress oncogenic Aurora A and induce autophagy by coadministrating curcumin with anticancer drugs for early-stage bladder cancer with high expression of Aurora A.

Suboptimal folic acid exposure rewires oncogenic metabolism and proteomics signatures to mediate human breast cancer malignancy.

Whether treatment with folic acid (FA) affects human breast cancer positively or negatively remains unclear. We subjected human Michigan Cancer Foundation-7 cells, a human breast cancer cell line, to suboptimal FA at low levels (10 nM; LF) and high levels (50 µM; HF) and investigated the molecular mechanisms underlying their effects through metabolic flux and systematic proteomics analyses. The data indicated that LF induced and HF aggravated 2-fold higher mitochondrial toxicity in terms of suppressed oxidative respiration, increased fermented glycolysis, and enhanced anchorage-independent oncospheroid formation. Quantitative proteomics and Gene Ontology enrichment analysis were used to profile LF- and HF-altered proteins involved in metabolism, apoptosis, and malignancy pathways. Through STRING analysis, we identified a connection network between LF- and HF-altered proteins with mammalian target of rapamycin (mTOR). Rapamycin-induced blockage of mTOR complex 1 (mTORC1) signaling, which regulates metabolism, differentially inhibited LF- and HF-modulated protein signatures of mitochondrial NADH dehydrogenase ubiquinone flavoprotein 2, mitochondrial glutathione peroxidase 4, kynureninase, and alpha-crystallin B chain as well as programmed cell death 5 in transcript levels; it subsequently diminished apoptosis and oncospheroid formation in LF/HF-exposed cells. Taken together, our data indicate that suboptimal FA treatment rewired oncogenic metabolism and mTORC1-mediated proteomics signatures to promote breast cancer development.

Vulnerability of Triple-Negative Breast Cancer to Saponin Formosanin C-Induced Ferroptosis.

Targeting ferritin via autophagy (ferritinophagy) to induce ferroptosis, an iron- and reactive oxygen species (ROS)-dependent cell death, provides novel strategies for cancer therapy. Using a ferroptosis-specific inhibitor and iron chelator, the vulnerability of triple-negative breast cancer (TNBC) MDA-MB-231 cells to ferroptosis was identified and compared to that of luminal A MCF-7 cells. Saponin formosanin C (FC) was revealed as a potent ferroptosis inducer characterized by superior induction in cytosolic and lipid ROS formation as well as GPX4 depletion in MDA-MB-231 cells. The FC-induced ferroptosis was paralleled by downregulation of ferroportin and xCT expressions. Immunoprecipitation and electron microscopy demonstrated the involvement of ferritinophagy in FC-treated MDA-MB-231 cells. The association of FC with ferroptosis was strengthened by the results that observed an enriched pathway with differentially expressed genes from FC-treated cells. FC sensitized cisplatin-induced ferroptosis in MDA-MB-231 cells. Through integrated analysis of differentially expressed genes and pathways using the METABRIC patients' database, we confirmed that autophagy and ferroptosis were discrepant between TNBC and luminal A and that TNBC was hypersensitive to ferroptosis. Our data suggest a therapeutic strategy by ferroptosis against TNBC, an aggressive subtype with a poor prognosis.

Virofree, an Herbal Medicine-Based Formula, Interrupts the Viral Infection of Delta and Omicron Variants of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) remains a threat with the emergence of new variants, especially Delta and Omicron, without specific effective therapeutic drugs. The infection causes dysregulation of the immune system with a cytokine storm that eventually leads to fatal acute respiratory distress syndrome (ARDS) and further irreversible pulmonary fibrosis. Therefore, the promising way to inhibit infection is to disrupt the binding and fusion between the viral spike and the host ACE2 receptor. A transcriptome-based drug screening platform has been developed for COVID-19 to explore the possibility and potential of the long-established drugs or herbal medicines to reverse the unique genetic signature of COVID-19. In silico analysis showed that Virofree, an herbal medicine, reversed the genetic signature of COVID-19 and ARDS. Biochemical validations showed that Virofree could disrupt the binding of wild-type and Delta-variant spike proteins to ACE2 and its syncytial formation via cell-based pseudo-typed viral assays, as well as suppress binding between several variant recombinant spikes to ACE2, especially Delta and Omicron. Additionally, Virofree elevated miR-148b-5p levels, inhibited the main protease of SARS-CoV-2 (Mpro), and reduced LPS-induced TNF-α release. Virofree also prevented cellular iron accumulation leading to ferroptosis which occurs in SARS-CoV-2 patients. Furthermore, Virofree was able to reduce pulmonary fibrosis-related protein expression levels in vitro. In conclusion, Virofree was repurposed as a potential herbal medicine to combat COVID-19. This study highlights the inhibitory effect of Virofree on the entry of Delta and Omicron variants of SARS-CoV-2, which have not had any effective treatments during the emergence of the new variants spreading.

Curcumin-induced mitotic spindle defect and cell cycle arrest in human bladder cancer cells occurs partly through inhibition of aurora A.

Curcumin, an active compound in turmeric and curry, has been proven to induce tumor apoptosis and inhibit tumor proliferation, invasion, angiogenesis, and metastasis via modulating numerous targets in various types of cancer cells. Aurora A is a mitosis-related serine-threonine kinase and plays important roles in diverse human cancers. However, the effect of curcumin on Aurora A has not been reported. In this study, Aurora A promoter activity and mRNA expression were inhibited in curcumin-treated human bladder cancer T24 cells, suggesting that Aurora A is regulated at the transcription level. We also found that curcumin preferentially inhibited the growth of T24 cells, which show a higher proliferation rate, invasion activity, and expression level of Aurora A compared with that of human immortalized uroepithelial E7cells. Furthermore, inhibition of phosphorylation of Aurora A and its downstream target histone H3 accompanied by the formation of monopolar spindle, induction of G(2)/M phase arrest, and reduction in cell division in response to curcumin were detected in T24 cells. These curcumin-induced phenomena were similar to those using Aurora A small interfering RNA and were attenuated by ectopic expression of Aurora A. Therefore, the antitumor mechanism of curcumin is Aurora A-related, which further supports the application of curcumin in treatments of human cancers.

Kinase gene expression and subcellular protein expression pattern of protein kinase C isoforms in curcumin-treated human hepatocellular carcinoma Hep 3B cells.

Curcumin, a yellow component of turmeric or curry powder, has been demonstrated to exhibit anti-carcinogenic effects in vitro, in vivo, and in human clinical trials. One of its molecular targets is protein kinase C (PKC) which has been reported to play essential roles in apoptosis, cell proliferation, and carcinogenesis. In this study, PKC mRNA expression was significantly inhibited in curcumin-treated human hepatocellular carcinoma (HCC) Hep 3B cells identified using a kinase cDNA microarray. Furthermore, curcumin decreased total protein expression of all PKCs in a time-related manner by immunoblotting of whole cell lysates, nuclear, membrane, and cytosolic fractions. In cytosolic fraction, the expression of PKC-α was totally inhibited by curcumin. In contrast, the expression levels of PKC-ζ and -µ were dramatically increased. Increases in expression of PKC-δ and PKC-ζ in the membrane and nucleus, and PKC-ι in the membrane were detected. In summary, the changes in expression and distribution of subcellular PKC isoforms in curcumin-treated Hep 3B cells suggest possible PKC-associated anti-tumor mechanisms of curcumin and provide alternative therapies for human HCC.

Saponin Formosanin C-induced Ferritinophagy and Ferroptosis in Human Hepatocellular Carcinoma Cells.

Ferroptosis, a recently discovered form of iron-dependent cell death, requires an increased level of lipid-reactive oxygen species (ROS). Ferritinophagy, a ferritin degradation pathway, depends on a selective autophagic cargo receptor (NCOA4). By screening various types of natural compounds, formosanin C (FC) was identified as a novel ferroptosis inducer, characterized by attenuations of FC-induced viability inhibition and lipid ROS formation in the presence of ferroptosis inhibitor. FC also induced autophagic flux, evidenced by preventing autophagic marker LC3-II degradation and increasing yellow LC3 puncta in tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) transfected cells when combined with autophagic flux inhibitor. It is noteworthy that FC-induced ferroptosis and autophagic flux were stronger in HepG2 cells expressing higher NCOA4 and lower ferritin heavy chain 1 (FTH1) levels, agreeing with the results of gene expression analysis using CTRP and PRISM, indicating that FTH1 expression level exhibited a significant negative correlation with the sensitivity of the cells to a ferroptosis inducer. Confocal and electron microscopy confirmed the pronounced involvement of ferritinophagy in FC-induced ferroptosis in the cells with elevated NCOA4. Since ferroptosis is a non-apoptotic form of cell death, our data suggest FC has chemotherapeutic potential against apoptosis-resistant HCC with a higher NCOA4 expression via ferritinophagy.

Pterostilbene Sensitizes Cisplatin-Resistant Human Bladder Cancer Cells with Oncogenic HRAS.

Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.