title: Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis.
abstract: OBJECTIVE
  Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells.
  
  
  METHODS
  Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5-5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot.
  
  
  RESULTS
  Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced.
  
  
  CONCLUSIONS
  Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid.
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Jintao
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Man
authors.initials: M
authors.lastname: Yi
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Longying
authors.initials: L
authors.lastname: Zha
authors.affiliation: Department of Certification Supervision, Guangdong Entry-Exit Inspection and Quarantine Bureau, Guojian Building, No.66, Huacheng Avenue, Zhujiang Xincheng, Guangzhou, Guangdong Province, P.R. China 510623.
authors.forename: Siqiang
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Zhijia
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Cheng
authors.initials: C
authors.lastname: Li
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Mingxing
authors.initials: M
authors.lastname: Gong
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Hong
authors.initials: H
authors.lastname: Deng
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Xinwei
authors.initials: X
authors.lastname: Chu
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Jiehua
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Zheqing
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Limei
authors.initials: L
authors.lastname: Mao
authors.affiliation: Department of Nutrition and Food Hygiene, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, No.1023 South Sha-Tai Rd, Guangzhou, Guangdong, P.R.China, 510515.
authors.forename: Suxia
authors.initials: S
authors.lastname: Sun
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D015153:Blotting, Western; D020148:Butyric Acid / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured
chemical_list: D012333:RNA, Messenger; D020148:Butyric Acid