Ruby L.Y. Chan

Host Immune Defense Peptide LL-37 Activates Caspase-Independent Apoptosis and Suppresses Colon Cancer

Cathelicidins are a family of bacteriocidal polypeptides secreted by macrophages and polymorphonuclear leukocytes (PMN). LL-37, the only human cathelicidin, has been implicated in tumorigenesis, but there has been limited investigation of its expression and function in cancer. Here, we report that LL-37 activates a p53-mediated, caspase-independent apoptotic cascade that contributes to suppression of colon cancer. LL-37 was expressed strongly in normal colon mucosa but downregulated in colon cancer tissues, where in both settings its expression correlated with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells. Exposure of colon cancer cells to LL-37 induced phosphatidylserine externalization and DNA fragmentation in a manner independent of caspase activation. Apoptogenic function was mediated by nuclear translocation of the proapoptotic factors, apoptosis-inducing factor (AIF) and endonuclease G (EndoG), through p53-dependent upregulation of Bax and Bak and downregulation of Bcl-2 via a pertussis toxin-sensitive G-protein-coupled receptor (GPCR) pathway. Correspondingly, colonic mucosa of cathelicidin-deficient mice exhibited reduced expression of p53, Bax, and Bak and increased expression of Bcl-2 together with a lower basal level of apoptosis. Cathelicidin-deficient mice exhibited an increased susceptibility to azoxymethane-induced colon tumorigenesis, establishing pathophysiologic relevance in colon cancer. Collectively, our findings show that LL-37 activates a GPCR-p53-Bax/Bak/Bcl-2 signaling cascade that triggers AIF/EndoG-mediated apoptosis in colon cancer cells.

FK-16 Derived from the Anticancer Peptide LL-37 Induces Caspase-Independent Apoptosis and Autophagic Cell Death in Colon Cancer Cells

Host immune peptides, including cathelicidins, have been reported to possess anticancer properties. We previously reported that LL-37, the only cathelicidin in humans, suppresses the development of colon cancer. In this study, the potential anticancer effect of FK-16, a fragment of LL-37 corresponding to residues 17 to 32, on cultured colon cancer cells was evaluated. FK-16 induced a unique pattern of cell death, marked by concurrent activation of caspase-independent apoptosis and autophagy. The former was mediated by the nuclear translocation of AIF and EndoG whereas the latter was characterized by enhanced expression of LC3-I/II, Atg5 and Atg7 and increased formation of LC3-positive autophagosomes. Knockdown of Atg5 or Atg7 attenuated the cytotoxicity of FK-16, indicating FK-16-induced autophagy was pro-death in nature. Mechanistically, FK-16 activated nuclear p53 to upregulate Bax and downregulate Bcl-2. Knockdown of p53, genetic ablation of Bax, or overexpression of Bcl-2 reversed FK-16-induced apoptosis and autophagy. Importantly, abolition of AIF/EndoG-dependent apoptosis enhanced FK-16-induced autophagy while abolition of autophagy augmented FK-16-induced AIF−/EndoG-dependent apoptosis. Collectively, FK-16 induces caspase-independent apoptosis and autophagy through the common p53-Bcl-2/Bax cascade in colon cancer cells. Our study also uncovered previously unknown reciprocal regulation between these two cell death pathways.

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.

Cigarette smoking and gastrointestinal diseases: The causal relationship and underlying molecular mechanisms (Review)

Cigarette smoking is an important risk factor for gastrointestinal (GI) disorders, including peptic ulcers, inflammatory bowel diseases, such as Crohns disease and cancer. In this review, the relationship between smoking and GI disorders and the underlying mechanisms are discussed. It has been demonstrated that cigarette smoking is positively associated with the pathogenesis of peptic ulcers and the delay of ulcer healing. Mechanistic studies have shown that cigarette smoke and its active ingredients can cause mucosal cell death, inhibit cell renewal, decrease blood flow in the GI mucosa and interfere with the mucosal immune system. Cigarette smoking is also an independent risk factor for various types of cancer of the GI tract. In this review, we also summarize the mechanisms through which cigarette smoking induces tumorigenesis and promotes the development of cancer in various sections of the GI tract. These mechanisms include the activation of nicotinic acetylcholine receptors, the formation of DNA adducts, the stimulation of tumor angiogenesis and the modulation of immune responses in the GI mucosa. A full understanding of these pathogenic mechanisms may help us to develop more effective therapies for GI disorders in the future.

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.

Effects of Cigarette Smoke and its Active Components on Ulcer Formation and Healing in the Gastrointestinal Mucosa

Ulceration in the gastrointestinal (GI) mucosa is a common disorder in humans. It has been shown that cigarette smoking is closely related to the increase of peptic ulcer and also plays an inhibitory role on ulcer healing. However, the underlying mechanisms by which cigarette smoke exerts these adverse effects remain largely unknown. It is perhaps partly due to the complexity of chemical compositions in the smoke and furthermore their pathological actions are largely undefined. In this review, we have highlighted the potential adverse effects of the toxic chemical components in cigarette smoke and summarized their possible mechanisms of actions on ulcer formation and healing in the GI tract. We also discuss in detail how cigarette smoke disturbs cell proliferation, influences mucus synthesis and secretion, delays blood vessel formation, and interferes the innate immune responses during ulceration and repair in the GI mucosa.

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.

Dihydrotanshinone induces p53-independent but ROS-dependent apoptosis in colon cancer cells

AIMS The therapeutic potential of various tanshinones was examined and compared for their anti-cancer activities on colon cancer cells. The role of ROS generation in the pro-apoptotic activity of dihydrotanshinone (DHTS) was further studied. MAIN METHODS Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis and poly-ADP-ribose-polymerase (PARP) cleavage were respectively measured by flow cytometer and Western blot. Changes of mitochondrial membrane potential (MMP), mitochondrial ROS (mitoROS) and total ROS were determined by confocal system under an inverted microscope. KEY FINDINGS Among the different tanshinones examined, DHTS produced the most potent anti-cancer effect. DHTS induced a selective cytotoxicity and apoptosis in both HCT116 p53(-/-) and HCT116 p53(+/+) colon cancer cells. A time- and concentration-dependent PARP cleavage further confirmed the apoptotic activity. In this regard, it was found DHTS provoked mitochondrial dysfunction in the early stage by decreasing MMP and mitoROS levels. This was followed by a time-dependent increase in intracellular ROS generation. Pretreatment with N-acetyl-l-cysteine (NAC) or catalase-PEG, the free radical scavengers, reduced apoptotic cell death. From these findings, it seems that leakage of ROS from mitochondria into cytosol by DHTS represents the major contributory factor leading to cell death in colon cancer cells. SIGNIFICANCE We report for the first time that DHTS induces apoptosis in colon cancer cells through a p53-independent pathway. Disturbance of ROS generation at the oxidative phosphorylation (OXPHOS) complex in mitochondria followed by the decrease of MMP and increase of intracellular ROS accumulation are suggested to be involved in the pro-apoptotic activity of DHTS.

Cathelicidin protects against Helicobacter pylori colonization and the associated gastritis in mice.

Cathelicidin, an antimicrobial peptide of the innate immune system, has been shown to modulate microbial growth, wound healing and inflammation. However, whether cathelicidin controls Helicobacter pylori infection in vivo remains unexplored. This study sought to elucidate the role of endogenous and exogenous mouse cathelicidin (CRAMP) in the protection against H. pylori infection and the associated gastritis in mice. Results showed that genetic ablation of CRAMP in mice significantly increased the susceptibility of H. pylori colonization and the associated gastritis as compared with the wild-type control. Furthermore, replenishment with exogenous CRAMP, delivered via a bioengineered CRAMP-secreting strain of Lactococcus lactis, reduced H. pylori density in the stomach as well as the associated inflammatory cell infiltration and cytokine production. Collectively, these findings indicate that cathelicidin protects against H. pylori infection and its associated gastritis in vivo. Our study also demonstrates the feasibility of using the transformed food-grade bacteria to deliver cathelicidin, which may have potential clinical applications in the treatment of H. pylori infection in humans.

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.