Aglycon of Rhizochalin from the Rhizochalina incrustata Induces Apoptosis via Activation of AMP-Activated Protein Kinase in HT-29 Colon Cancer Cells

Abstract

Rhizocalin is the first two-headed sphingolipid like compound isolated from the sponge Rhizochalina incrustata.1) It has been shown that rhizochalin and its derivatives have antibacterial, selective DNA-damaging, antifungal and cytotoxic activity.1) In addition, accumulating evidence suggests that rhizochalin and its derivatives have potent antitumor activity in human cancer cell lines, including cervical cancer, leukemia, and colon cancer.2,3) However, the underlying mechanisms of its anticarcinogenic activity remain poorly understood.The AMP-activated protein kinase (AMPK), a serine/ theronine kinase present in all eukaryotes, is a sensor of cellular energy status. 4) Since tumor-suppressor genes such as TSC2 and LKB1 function as upstream kinases of AMPK, AMPK is known as an antiproliferative molecule. 5,6) Previous studies have reported that AMPK activators, 5-amino-4-imidazolecarboxamide riboside (AICAR) and metformin, inhibit the cell growth in cultured tumor cells and mouse xenograft models.6-8) AMPK activation also induces apoptosis in cancer cell lines such as neuroblastoma cells, pancreatic cells, glioma cells, and endometrial cancer cells. [9][10][11][12] In addition, AMPK activation by phytochemicals, such as quercetin and 24-hydroxyursolic acid, is involved in apoptosis in colon cancer cell lines 13,14) Therefore, AMPK activation by naturally occurring compounds has attractive potentials for cancer therapy.Here, we have investigated the role of aglycon of rhizochalin (AglRhz), two-headed sphingolipid-like compound isolated from the sponge Rhizochalina incrustata, in the activation of AMPK and induction of apoptosis in HT-29 colon cancer cells. We observed that AglRhz inhibited phosphorylation of p70S6K and its downstream target extracellular signal-regulated kinase (ERK) in a Raptor-dependent manner via AMPK activation. We have also found that AglRhz induced the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), as well as DNA fragmentation, resulting in the induction of apoptosis. In addition, AglRhz inhibited insulin-like growth factor (IGF)-induced cell neoplastic transformation in JB6 Cl41 cells and tumorigenicity in HT-29 cells. Taken together, our findings illustrate that AglRhz has a potent antitumor activity against the HT-29 colon cancer cells and suggests a molecular mechanism that underlies the antitumor activity of AglRhz. MATERIALS AND METHODS Reagents and AntibodiesMcCoy's 5A medium, L-glutamine, gentamicin, and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Polyvinyl idene difluoride (PVDF) membrane was obtained from Millipore (Bedford, MA, U.S.A.). 3-[4,5-Dimethylthiazol-2-thiazoyl]-2,5-diphenyltetrazolium bromide (MTT) was from Sigma-Aldrich (St. Louis, MO, U.S.A.). Antibodies against phospho-p70S6K (Thr389), p70S6K, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-Raptor (Ser792), phospho-AMPK, cleaved caspase-3, cleaved PARP, caspase-3, and B-cell lymphoma 2 (Bcl-2) were from Cell Signaling Technology Inc. (Beverly, MA, U.S.A.); antibodies...

A Survey of Marine Natural Compounds and Their Derivatives with Anti-Cancer Activity Reported in 2011

“…AMPK phosphorylation by AglRhz results in the inhibition of AP-1 via a cascade of biochemical reactions. This finding was confirmed by the significant inhibition of IGF-I-induced neoplastic cell transformation of JB6 Cl 41 cells [ 79 ]. The chemical structure of AglRhz shows the presence of a ketone moiety in the middle of the backbone and amine and hydroxyl moieties at each head ( 23 ).…”

“…Rhizochalin is a two-headed sphingolipid-like molecule that was isolated from the marine sponge Rhizochalina incrustata [ 78 , 79 ]. Several analogs of this compound were either isolated or synthetized since its discovery in 1989 [ 78 ].…”

“…Since 2009, the aglycon of rhizochalin (AglRhz) was found to be cytotoxic to many cancer cell lines, such as uterine, cervical and colon cancers as well as leukemia [ 80 , 81 ]. Its mechanism of action was elucidated in 2011 through the work of Khanal et al , who demonstrated that AglRhz induces AMPK, caspase-3 and PARP activation as well as DNA fragmentation in HT-29 cells [ 79 ]. All of these effects result in apoptosis and suppression of the tumorigenicity of HT-29 cells.…”

“…All of these effects result in apoptosis and suppression of the tumorigenicity of HT-29 cells. Moreover, AglRhz inhibits insulin-like growth factor (IGF)-1-induced AP-1 activity and cellular transformation in JB6 Cl 41 cells [ 79 ]. It is known that AMPK has a role in metabolism and cell growth regulation and thus has become a target in cancer therapy.…”

Synthesis and anticancer activity of the derivatives of marine compound rhizochalin in castration resistant prostate cancer

“…Analyses of structure-activity relationships and further structure optimization are essential steps in the drug development process. For rhizochalin (1) (Figure 1 ) [ 13 ] and similar compounds it has been described that sugar elimination may lead to the increased pro-apoptotic activity [ 14 – 16 ]. The change in the molecule polarity due to an additional reduction of the keto group, or the introduction of nitrogen in the molecule may also affect the biological activity, including pro-apoptotic activity of the drug, as well its effect on autophagy [ 17 ].…”