Jing Shen

Reversal of P-glycoprotein (P-gp) mediated multidrug resistance in colon cancer cells by cryptotanshinone and dihydrotanshinone of Salvia miltiorrhiza.

OBJECTIVE Multidrug resistance (MDR) of cancer cells to a broad spectrum of anticancer drugs is an obstacle to successful chemotherapy. Overexpression of P-glycoprotein (P-gp), an ATP-binding cassette (ABC) membrane transporter, can mediate the efflux of cytotoxic drugs out of cancer cells, leading to MDR and chemotherapy failure. Thus, development of safe and effective P-gp inhibitors plays an important role in circumvention of MDR. This study investigated the reversal of P-gp mediated multidrug resistance in colon cancer cells by five tanshinones including tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone and miltirone isolated from Salvia miltiorrhiza (Danshen), known to be safe in traditional Chinese medicine. METHODS The inhibitory effects of tanshinones on P-gp function were compared using digoxin bi-directional transport assay in Caco-2 cells. The potentiation of cytotoxicity of anticancer drugs by effective tanshinones were evaluated by MTT assay. Doxorubicin efflux assay by flow cytometry, P-gp protein expression by western blot analysis, immunofluorescence for P-gp by confocal microscopy, quantitative real-time PCR and P-gp ATPase activity assay were used to study the possible underlying mechanisms of action of effective tanshinones. RESULTS Bi-directional transport assay showed that only cryptotanshinone and dihydrotanshinone decreased digoxin efflux ratio in a concentration-dependent manner, indicating their inhibitory effects on P-gp function; whereas, tanshinone I, tanshinone IIA and miltirone had no inhibitory effects. Moreover, both cryptotanshinone and dihydrotanshinone could potentiate the cytotoxicity of doxorubicin and irinotecan in P-gp overexpressing SW620 Ad300 colon cancer cells. Results from mechanistic studies revealed that these two tanshinones increased intracellular accumulation of the P-gp substrate anticancer drugs, presumably by down-regulating P-gp mRNA and protein levels, and inhibiting P-gp ATPase activity. CONCLUSIONS Taken together, these findings suggest that cryptotanshinone and dihydrotanshinone could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies for colon cancer.

Sensitivity of apoptosis-resistant colon cancer cells to tanshinones is mediated by autophagic cell death and p53-independent cytotoxicity.

BACKGROUND Multidrug resistance (MDR) develops in nearly all patients with colon cancer. The reversal of MDR plays an important role in the success of colon cancer chemotherapy. One of the commonest mechanisms conferring MDR is the suppression of apoptosis in cancer cells. PURPOSE This study investigated the sensitivity of cryptotanshinone (CTS) and dihydrotanshinone (DTS), two lipophilic tanshinones from a traditional Chinese medicine Salvia miltiorrhiza, in apoptosis-resistant colon cancer cells. METHODS Cell viability was measured by MTT assay. Cell cycle distribution and apoptosis were determined by flow cytometry. Protein levels were analyzed by western blot analysis. The formation of acidic vesicular organelles was visualized by acridine orange staining. RESULTS Experimental results showed that multidrug-resistant colon cancer cells SW620 Ad300 were sensitive to both CTS and DTS in terms of cell death, but with less induction of apoptosis when compared with the parental cells SW620, suggesting that other types of cell death such as autophagy could occur. Indeed, the two tanshinones induced more LC3B-II accumulation in SW620 Ad300 cells with increased autophagic flux. More importantly, cell viability was increased after autophagy inhibition, indicating that autophagy induced by the two tanshinones was pro-cell death. Besides, the cytotoxic actions of the two tanshinones were p53-independent, which could be useful in inhibiting the growth of apoptosis-resistant cancer cells with p53 defects. CONCLUSION The current findings strongly indicate that both CTS and DTS could inhibit the growth of apoptosis-resistant colon cancer cells through induction of autophagic cell death and p53-independent cytotoxicity. They are promising candidates to be further developed as therapeutic agents in the adjuvant therapy for colon cancer, especially for the apoptosis-resistant cancer types.

Dihydrotanshinone I induced apoptosis and autophagy through caspase dependent pathway in colon cancer.

BACKGROUND Dihydrotanshinone I (DHTS) was previously reported to exhibit the most potent anti-cancer activity among several tanshinones in colon cancer cells. Its cytotoxic action was reactive oxygen species (ROS) dependent but p53 independent. PURPOSE To further study the anti-cancer activity of DHTS and its molecular mechanisms of action in colon cancer both in vitro and in vivo. METHODS Caspase activity was detected by fluorescence assay. Apoptosis was detected by flow cytometry and TUNEL assay. Protein levels were analyzed by western blotting. Knockdown of target gene was achieved by siRNA transfection. Formation of LC3B puncta and activation of caspase-3 were detected by confocal fluorescence microscope. In vivo anti-colon cancer activity of DHTS was observed in xenograft tumors in NOD/SCID mice. RESULTS Anti-colon cancer activity of DHTS by inducing apoptosis and autophagy was observed both in vitro and in vivo. Mitochondria mediated caspase dependent pathway was essential in DHTS-induced cytotoxicity. The apoptosis induced by DHTS was suppressed by knockdown of apoptosis inducing factor (AIF), inhibition of caspase-3/9 but was increased after knockdown of caspase-2. Meantime, knockdown of caspase-2, pretreatment with Z-VAD-fmk or NAC (N-Acety-L-Cysteine) efficiently inhibited the autophagy induced by DHTS. A crosstalk between cytochrome c and AIF was also reported. CONCLUSION DHTS-induced caspase and ROS dependent apoptosis and autophagy were mediated by mitochondria in colon cancer. DHTS could be a promising leading compound for the development of anti-tumor agent or be developed as an adjuvant drug for colon cancer therapy.

Miltirone induced mitochondrial dysfunction and ROS-dependent apoptosis in colon cancer cells

AIMS To study the characteristics of miltirone-induced anti-colon cancer effects. MATERIALS AND METHODS Cell viability was detected using MTT assay. LDH (lactate dehydrogenase) leakage was detected using CytoTox96® non-radioactive cytotoxicity kit. Apoptosis was detected by FCM (flow cytometry). Caspase activation was determined by chemiluminescence or western blotting. AIF (apoptosis-inducing factor) expression in the cell fraction was determined by western blotting. ROS (reactive oxygen species), MMP (mitochondrial membrane potential) and mitochondrial mass were determined by confocal microscope. Intracellular calcium was detected by both FCM and confocal microscope. To determine the roles of ROS and Ca(2+) in the pro-apoptotic activity of miltirone, colon cancer cells were pretreated with kinds of antioxidants, dicoumarol, calpeptin or BAPTA-AM in some cases. KEY FINDINGS Miltirone exhibited potent cytotoxicity on colon cancer cells with a better selectivity than that of dihydrotanshinone. The pro-apoptotic activity of miltirone was p53- and ROS-dependent. In detail, miltirone induced direct mitochondrial damage, including significant decrease of mitochondrial ROS, MMP, mass and increase of intracellular ROS and Ca(2+). NQO1 (quinone oxidoreductase1) was supposed to be a defender for the cytotoxicity induced by miltirone in colon cancer cells. Furthermore, miltirone induced time- and concentration-dependent translocation of AIF and activation of caspases. SIGNIFICANCE In this study, ROS- and p53-dependent apoptosis induced by miltirone on colon cancer cells was firstly revealed. Strong positive feedback between mitochondrial dysfunction and accumulation of intracellular Ca(2+) was suggested to be the characteristic of the anti-colon cancer activity of miltirone.

Vascular-targeted TNFα and IFNγ inhibits orthotopic colorectal tumor growth

BackgroundTumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) were originally identified to show potent anti-tumor activity and immunomodulatory capability. Unfortunately, several clinical studies of relevant cancer therapy did not observe significant response in maximum tolerated dose whether given alone or in combination. We have identified a tumor vasculature homing peptide (TCP-1 peptide) which targets only the vasculature of colorectal tumors but not normal blood vessels in animals and humans. In the current study, the antitumor effect of TCP-1/TNFα and TCP-1/IFNγ alone or in combination was studied in orthotopic colorectal tumor model.MethodsTCP-1/TNFα and TCP-1/IFNγ recombinant proteins were prepared and i.v. injected to study the in vivo anticancer effect in orthotopic colorectal tumor model. Tumor apoptosis was determined by TUNEL staining and cleaved caspase-3 immunofluorescent staining. Tumor infiltrating lymphocytes were analyzed by immunofluorescent staining and flow cytometry. Western-blot was performed to examine the expression of proteins. Cell apoptosis was measured by Annexin V/PI flow cytometry.ResultsTargeted delivery of TNFα or IFNγ by TCP-1 peptide exhibited better antitumor activity than unconjugated format by inducing more tumor apoptosis and also enhancing antitumor immunity shown by increased infiltration of T lymphocytes inside the tumor. More importantly, combination therapy of TCP-1/TNFα and TCP-1/IFNγ synergistically suppressed tumor growth and alleviated systematic toxicity associated with untargeted therapy. This combination therapy induced massive apoptosis/secondary necrosis in the tumor.ConclusionsTaken together, our data demonstrate TCP-1 is an efficient drug carrier for targeted therapy of colorectal cancer (CRC). TCP-1/TNFα combined with TCP-1/IFNγ is a promising combination therapy for CRC.

Comprehensive molecular profiling of the B7 family of immune-regulatory ligands in breast cancer

ABSTRACT The B7 gene family has crucial roles in the regulation of adaptive cellular immunity. In cancer, deregulation of co-inhibitory B7 molecules is associated with reduced antitumor immunity and cancer immune evasion. FDA approval of cancer immunotherapy antibodies against cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death-1 (PD-1)—both ligands of the B7 family—demonstrate the impact of these checkpoint regulators in cancer. Using data from cBioPortal, we performed comprehensive molecular profiling of the 10 currently known B7 family proteins in 105 different cancers. B7 family members were amplified in breast cancer: with B7 mRNA levels upregulated in a cohort of 1,098 patients with all types of breast cancer and in 82 patients with triple-negative breast cancer. Promoter methylation analysis indicated an epigenetic basis for deregulation of certain B7 family genes in breast cancer. Moreover, patients with B7-H6 genomic alterations had significantly worse overall survival, and certain clinical attributes were associated with B7-H6 expression, which indicates that B7-H6 may be a potential target for breast cancer immunotherapy. Finally, using network analysis (based on data from cBioportal), we identified BTLA, MARCH8, PLSCR1 and SMAD3 as potentially involved in T cell signaling under regulation of B7 family proteins.

Critical Role of Antimicrobial Peptide Cathelicidin for Controlling Helicobacter pylori Survival and Infection

The antimicrobial peptide cathelicidin is critical for protection against different kinds of microbial infection. This study sought to elucidate the protective action of cathelicidin against Helicobacter pylori infection and its associated gastritis. Exogenous cathelicidin was found to inhibit H. pylori growth, destroy the bacteria biofilm, and induce morphological alterations in H. pylori membrane. Additionally, knockdown of endogenous cathelicidin in human gastric epithelial HFE-145 cells markedly increased the intracellular survival of H. pylori. Consistently, cathelicidin knockout mice exhibited stronger H. pylori colonization, higher expression of proinflammatory cytokines IL-6, IL-1β, and ICAM1, and lower expression of the anti-inflammatory cytokine IL-10 in the gastric mucosa upon H. pylori infection. In wild-type mice, H. pylori infection also stimulated gastric epithelium-derived cathelicidin production. Importantly, pretreatment with bioengineered Lactococcus lactis that actively secretes cathelicidin significantly increased mucosal cathelicidin levels and reduced H. pylori infection and the associated inflammation. Moreover, cathelicidin strengthened the barrier function of gastric mucosa by stimulating mucus synthesis. Collectively, these findings indicate that cathelicidin plays a significant role as a potential natural antibiotic for H. pylori clearance and a therapeutic agent for chronic gastritis.

Therapeutic targeting of noncoding RNAs in hepatocellular carcinoma: Recent progress and future prospects (Review)

Due to the high mortality rate and unsatisfactory treatment options available, hepatocellular carcinoma (HCC) remains one of the most common malignancies and a leading cause of cancer-associated mortality. Novel therapeutic targets for HCC are urgently required. Advanced RNA sequencing technology enables the identification of considerable amounts of noncoding RNAs (ncRNAs), including small noncoding RNAs and long noncoding RNAs, which exhibit no protein-coding activities. In this respect, ncRNAs and their regulatory processes are important factors in liver tumorigenesis. The present review focuses on the characteristics and biological roles of ncRNAs in HCC. Potential therapeutic applications of ncRNAs in HCC are also evaluated.

The Beneficial Effects of Quercetin, Curcumin, and Resveratrol in Obesity

Over the past two decades, obesity has been one of the major public health concerns in most countries. In the search for new molecules that could be used for the treatment of obesity, good perspectives have been opened up for polyphenols, a class of natural bioactive phytochemicals. Experimental and limited clinical trial evidence supports that some polyphenols such as quercetin, curcumin, and resveratrol have potential benefit functions on obesity treatment. This brief review focuses on the main functions of the above-named polyphenols on adipose tissue. These polyphenols may play beneficial effects on adipose tissue under obese condition by alleviating intracellular oxidative stress, reducing chronic low-grade inflammation, inhibiting adipogenesis and lipogenesis, and suppressing the differentiation of preadipocytes to mature adipocytes.

1,25-Dihydroxyvitamin D3 suppresses gastric cancer cell growth through VDR- and mutant p53-mediated induction of p21

Aims: Previous studies have indicated that vitamin D deficiency correlates with cancer risk and vitamin D potentiates antitumor effects in a variety of cancers. The antitumor effect of vitamin D on gastric cancer was rarely studied. We aimed to investigate the antitumor effect of vitamin D on gastric cancer and underlying mechanisms. Main methods: We investigated the antitumor activity of the active form of vitamin D, 1&agr;,25‐dihydroxyvitamin D3 (1,25(OH)2D3) on gastric cancer cells (TMK1) and immortalized normal gastric cells (HFE145) by using MTT, colony formation and Flow cytometry assays. We demonstrated the important role of vitamin D receptor (VDR) and mutant p53 (mutp53) in mediating the antitumor action of 1,25(OH)2D3 by using siRNA, western‐blot, immunofluorescent staining and immunoprecipitation assays. Key findings: 1,25(OH)2D3 could significantly inhibit proliferation and induce cell cycle arrest in TMK1 but not in HFE145. Furthermore, 1,25(OH)2D3 stimulated p21 expression and suppressed cyclin‐dependent kinase 2 (CDK2) expression in TMK1 in a VDR‐dependent manner. High levels of VDR in human gastric cancer tissues and cancer cell lines implicated that vitamin D could display more potent pharmacological action against malignant cells. Besides, mutp53 but not wild type p53 was essential for 1,25(OH)2D3‐stimulated upregulation of p21 in gastric cancer cells. Indeed, mutp53 could stabilize nuclear VDR level through interaction with VDR. Significance: Our results suggest that 1,25(OH)2D3 inhibits gastric cancer cell growth through VDR and mutp53 interaction followed by the modulation of p21/CDK2. We propose that vitamin D might be used for the prophylactic treatment for malignant diseases in the stomach.