A [Lys⁴⁹]phospholipase A₂fromProtobothrops flavoviridisVenom Induces Caspase-Independent Apoptotic Cell Death Accompanied by Rapid Plasma-Membrane Rupture in Human Leukemia Cells

Abstract

]PLA 2 that has much stronger lipolytic activity than BPII, failed to induce cell death. BPIItreated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPIIinduced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive pbromophenacylated BPII induced cell death, BPIIinduced apoptotic cell death is independent of PLA 2 lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.Key words: Protobothrops flavoviridis; [Lys 49 ]phospholipase A 2 ; caspase-independent cell death Snake venoms are complex mixtures of a number of physiologically active proteins including metalloproteases, 1,2) thrombin-like enzymes, 3,4) complement activating enzyme, 5) phospholipases A 2 (PLA 2 s) [EC 3.1.1.4] 6,7) and so on. PLA 2 catalyzes the hydrolysis of 1,2-diacyl-3-sn-phosphoglycerides at the C2-position with a requirement for Ca 2þ . 8) PLA 2 s are abundant in the mammalian pancreas and also in reptile and insect venoms. Snake venoms contain plural PLA 2 isozymes, which show a wide variety of physiological activities, including induction of edema, 9) neurotoxicity, 10) and myotoxic activity.11) Snake-venom PLA 2 isozymes are classified into groups I and II based on the mode of disulfide pairing. 13,14) It is well established that these PLA 2 isozymes have the same scaffold. 15,16) Protobothrops (formerly Trimeresurus) flavoviridis (Habu snake, Crotalinae) inhabit southwestern islands of Japan, Amami-Oshima, Tokunoshima, and Okinawa. Five PLA 2 isozymes have been isolated from P. flavoviridis (Amami-Oshima Island, Japan) venom. [17][18][19][20][21] They all consist of 122 amino acid residues and have been well studied: 22) It is known that BPII is 10-100 times more active than BPI in contracting activity in the guinea pig ileum and artery. 23) In the study reported here, we examined the cell death activity of P. flavoviridis venom PLA 2 isozymes BPII and PLA2. It was found that BPII can induce caspaseindependent cell death in human leukemia cells regardless of its depressed enzymatic activity, but PLA2, much more active and the most abundant [Asp 49 ]PLA 2 in P. flavoviridis venom, does not induce cell death. The characteristics and mechanism of cell death induced by BPII were investigated.y To whom correspondence should be addressed. Fax: +81-96-326-5048; E-mail: naoko@ph.sojo-u.ac.jp Abbreviations: Ac, acetyl; AcD, actinomycin D; AMC, 7-amino-4-methylcoumarin; pBPB, p-bromophenacyl bromide; CK, creatine kinase; CM, S-carboxymethyl; FITC, fluorescein isothiocyanate; fmk, fluoromethyl ketone; HRP, horseradish peroxidase; FSC, forward scatter; LDH, lactate dehydrogenase; OMe, methoxy; PARP, poly(ADP-ribo...

Propensity of crocin to offset Vipera russelli venom induced oxidative stress mediated neutrophil apoptosis: a biochemical insight

“…The interplay between oxidative stress and inflammation has been implicated in the pathophysiology of many diseases including cardiovascular diseases, diabetes, cancer, arthritis, chronic kidney and neurological diseases. Several studies have demonstrated that the snake venom and their isolated components such as PLA 2 and metalloproteases induce apoptosis in different cell types (Valko et al 2007;Brenes et al 2010;Murakami et al 2011;Park et al 2012). Venom induced cell death is currently the subject of considerable interest for toxicologists in order to understand the molecular mechanism and to explore the possible therapeutic purposes of venom.…”

“…Additionally, the results demonstrated in the present study may be correlated to VR venom induced pharmacological effects and could also contribute to the elucidation of possible mechanism of action of target specific toxins. Several SVMPs (VAP1, VAP2, VLAIP, graminelysin), L-amino acid oxidase and PLA 2 have been reported to induce apoptosis in HUVECs and several cancer cell lines including human liver (Bel7402), human leukemia (K-562) and human gastric carcinoma (BGC823) cells (Zhu et al 2010;Murakami et al 2011;Teklemariam et al 2011).…”

Patatin-like PLA<sub>2</sub> with cytotoxicity against mammalian and plant tumour cells

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