Clover Ching Man Wong

Inhibition of macroautophagy by bafilomycin A1 lowers proliferation and induces apoptosis in colon cancer cells.

Macroautophagy is a process by which cytoplasmic content and organelles are sequestered by double-membrane bound vesicles and subsequently delivered to lysosomes for degradation. Macroautophagy serves as a major intracellular pathway for protein degradation and as a pro-survival mechanism in time of stress by generating nutrients. In the present study, bafilomycin A(1), a vacuolar type H(+)-ATPase inhibitor, suppresses macroautophagy by preventing acidification of lysosomes in colon cancer cells. Diminished macroautophagy was evidenced by the accumulation of undegraded LC3 protein. Suppression of macroautophagy by bafilomycin A(1) induced G(0)/G(1) cell cycle arrest and apoptosis which were accompanied by the down-regulation of cyclin D(1) and cyclin E, the up-regulation of p21(Cip1) as well as cleavages of caspases-3, -7, -8, and -9 and PARP. Further investigation revealed that bafilomycin A(1) increased the phosphorylation of ERK, JNK, and p38. In this regard, p38 inhibitor partially reversed the anti-proliferative effect of bafilomycin A(1). To conclude, inhibition of macroautophagy by bafilomycin A(1) lowers G(1)-S transition and induces apoptosis in colon cancer cells. Our results not only indicate that inhibitors of macroautophagy may be used therapeutically to inhibit cancer growth, but also delineate the relationship between macroautophagy and apoptosis.

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.

2,3′,4,4′,5′‐Pentamethoxy‐trans‐stilbene, a resveratrol derivative, inhibits colitis‐associated colorectal carcinogenesis in mice

Background and purpose:  Resveratrol, a naturally occurring polyphenolic antioxidant, has been shown to exhibit chemoprophylactic effects on cancer development. Previously, we reported that 2,3′,4,4′,5′‐pentamethoxy‐trans‐stilbene (PMS), a methoxylated resveratrol derivative, exerted a highly potent anti‐proliferative effect on human colon cancer cells as compared with its parent compound. In the present study, the chemopreventive effect of PMS was evaluated in a mouse model of colitis‐associated colon carcinogenesis.

A novel peptide specifically targeting the vasculature of orthotopic colorectal cancer for imaging detection and drug delivery

Colorectal cancer (CRC) is the third most common malignancy and the fourth most frequent cause of cancer deaths worldwide. Ligand-mediated diagnosis and targeted therapy would have vital clinical applications in cancer treatment. In this study, an orthotopic model of colorectal cancer was established in mice. In vivo phage library selection was then utilized to isolate peptides specifically recognizing the vasculature of colorectal cancer tissues. A phage (termed TCP-1 phage) was isolated by this manner and it homed to the colorectal cancer tissues by 11- to 94-fold more than other organs. Chemical synthetic peptide (CTPSPFSHC, termed TCP-1) displayed by TCP-1 phage inhibited the homing ability of the phage to the tumor mass when co-injected intravenously with the TCP-1 phage into mice with colon cancer. Meanwhile, immunostaining analysis indicated that TCP-1 phage and peptide localized in the vasculature of the colorectal cancer tissue, but not of normal tissues. Moreover, TCP-1 peptide bound to blood vessels of surgical tissue samples of human colorectal cancer. After intravenous injection of FITC-labeled TCP-1 into the tumor-bearing mice for 20h, there was a strong fluorescent signal in the tumors but not other tissues when observed under blue light. In addition, TCP-1 conjugated with a pro-apoptotic peptide specifically induced apoptosis of tumor-associated blood vessels in vivo. The data define a novel peptide TCP-1 as an effective agent for imaging detection and drug delivery for colorectal cancer.

Indomethacin and SC236 enhance the cytotoxicity of doxorubicin in human hepatocellular carcinoma cells via inhibiting P-glycoprotein and MRP1 expression.

Doxorubicin is a chemotherapeutic drug widely used for the treatment of hepatocellular carcinoma but its efficacy is restricted by multidrug resistance. Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2-selective inhibitors exhibit anti-cancer properties as well as abilities to overcome drug resistance. In the present study, indomethacin (a NSAID) and SC236 (a COX-2-selective inhibitor) enhanced the cytotoxicity of doxorubicin in the hepatocellular carcinoma cell line HepG2 and its drug-resistant sub-line R-HepG2. Both drugs increased the intracellular accumulation and retention of doxorubicin in vitro. The effects were not reversed by prostaglandin E(2), implicating a COX-independent mechanism. Indomethacin and SC236 partially reversed the increase in expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) induced by doxorubicin in R-HepG2 cells. In conclusion, indomethacin and SC236 increased the intracellular accumulation and retention of doxorubicin and thus its cytotoxicity in HepG2 and drug-resistant HepG2 cells. These effects, mediated through decrease in P-gp and MRP1 expression and/or direct inhibition of P-gp activity, may improve multidrug resistant-cancer chemotherapy.

Protective effects of cathelicidin-encoding Lactococcus lactis in murine ulcerative colitis

Background and Aim:  Intrarectal administration of mouse cathelin‐related antimicrobial peptide (mCRAMP) reduced intestinal inflammation in mice. In the current study, we examined whether mCRAMP‐transformed Lactococcus lactis given orally attained similar protective effects.

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.

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.

Cathelicidins in inflammation and tissue repair: Potential therapeutic applications for gastrointestinal disorders.

AbstractCathelicidins, a family of host defense peptides, are highly expressed during infection, inflammation and wound healing. These peptides not only have broad-spectrum antimicrobial activities, but also modulate inflammation by altering cytokine response and chemoattraction of inflammatory cells in diseased tissues. In this connection, a mouse cathelicidin has been demonstrated to prevent inflammation in the colon through enhancing mucus production and reducing production of pro-inflammatory cytokines. In addition, cathelicidins promote wound healing through stimulation of re-epithelialization and angiogenesis at injured tissues. In an animal model of gastric ulceration, the rat cathelicidin promotes ulcer healing by inducing proliferation of gastric epithelial cells both in vitro and in vivo. In conclusion, cathelicidins represent an important group of effector molecules in the innate immune system that operates a complex integration of inflammation and tissue repair in the gastrointestinal mucosa and other organs.