p53‐independent anti‐tumor effects of the nitrogen‐containing bisphosphonate zoledronic acid

Abstract

Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, exerts anti-tumor effects by inhibiting the prenylation of small GTPases. We have also reported that ZOL shows an anti-leukemic effect by inducing apoptosis throughout the S phase to the G 2 /M boundary. Here, we studied the effects of ZOL on various cell cycle regulators, including p53, cyclin-dependent kinases (CDKs), CDK inhibitors and cyclins, using BV173 leukemia and HCT116 colorectal carcinoma cell lines, harboring wild-type (wt-) p53. ZOL induced the accumulation of neither p53 nor p21 WAF1/CIP1 during the execution of apoptosis in BV173 cells. Therefore, we investigated the dependence of ZOL-induced apoptosis on intact p53 by using wt-p53 HCT116 and a p53-degraded HCT116 subline, and observed no significant difference. ne particularly attractive approach in the development of more potent anticancer therapies involves the blockade of deregulated cellular signaling pathways. The use of imatinib mesylate, a competitive inhibitor of ABL tyrosine kinase, on Philadelphia-positive leukemias is the paradigm for this type of tumor-selective targeted therapy, 1) and gefitinib, a selective inhibitor of epidermal growth factor receptor-tyrosine kinase inhibitor, has shown promise in patients with solid tumors. 2)Because Ras signaling pathways are frequently overly active in leukemias and solid tumors, an approach that inhibits the posttranslational prenylation of small GTPases by farnesyl transferase inhibitors or nitrogen-containing bisphosphonates (BPs) is intriguing.3-6) One such nitrogen-containing BP is zoledronic acid (ZOL), and recent studies showed that it has in vitro antiproliferative potency against several types of cancer cell lines.4, 5) Also, in a previous study, we found that ZOL potentially induces apoptosis in leukemic cell lines and also prolongs the survival of mice xenografted with a leukemic cell line. 7)Anticancer drugs generally induce apoptosis by triggering biochemical events involved not only in apoptotic pathways, but also in cell cycle regulation. 8) To characterize the underlying mechanisms of apoptotic induction in cancer cells by ZOL, we assessed its effects on cell cycle regulators, including p53, one of the key regulators targeted by various anticancer agents. By testing cancer cell lines with wild-type (wt-) p53 and p53-degraded cell lines, we reveal for the first time that the anti-tumor effect of ZOL is not reduced by the loss of p53 function, although the contribution of p53 to the anti-tumor effect of

Zoledronic acid determines S-phase arrest but fails to induce apoptosis in cholangiocarcinoma cells

“…Taken together our results on phase distribution of cells, expression of cyclin proteins and their regulators pRb and p27 KIP1 suggest that ZOL treatment on cholangiocarcinoma cells do not M a n u s c r i p t 18 induce G1/S arrest, as already seen in other tumor cells [26,46]. It determines an arrest of cells in the S-phase without an accumulation of cells in the G2-phase and absence of apoptosis induction.…”

“…It should be noted that cells, exposed up to IC 50 ZOL for 72h, following drug removal later on resume their growth thus indicating the reversibility of the inhibition. In our opinion, this property of ZOL suggests its [23][24][25][26][27]. To date, however, the details of the molecular mechanism of cell cycle arrest involved remain obscure.…”

“…These are required for the posttranslational modification (prenylation) of small GTP proteins (such as Rho, Ras, Rac) that play crucial roles in signaling pathways controlling cell growth. The anti-proliferative effect of ZOL was described in several tumor cell lines and it was likely due to cell cycle distribution change by accumulating and arresting cells in the S-phase [23][24][25][26][27]. This arrest was coupled to changing levels of cyclins and cell cycle regulators, and the S-phase arrest was associated with typical (caspase-dependent) [25,27,28] or atypical (caspase-independent) apoptotic pathway.…”

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia

“…Total RNA was extracted by using the Micro-to-Midi Total RNA Extraction Kit (Invitrogen, San Diego, CA, USA) and subjected to reverse transcription. 40. The mRNA levels of human bad, bik, bim, bmf, hrk, noxa and puma were analyzed by using the LightCycler System (Roche Diagnostics, Sandhoferstrabe, Mannheim, Germany) with FastStart DNA Master SYBR Green I (Roche).…”

“…Caspase-8 activity was measured by using the caspase-8 fluorometric assay kit (MBL, Nagoya, Japan) and Wallac Victor 2 Multi-label Counters (Perkin Elmer, Wellesley, MA). 40 Combination cell killing effects with two agents from INNO-406, 17-AAG, PS-341 and ABT-737 on Bcr-Abl þ leukemic cells. Cells were treated with various concentrations of INNO-406, 17-AAG, PS-341 or ABT-737 alone or the combinations of two of listed agents at various concentrations as indicated in the figures.…”