Restriction of cisplatin induction of acute apoptosis to a subpopulation of cells in a three‐dimensional carcinoma culture model

Abstract

Cisplatin is a clinically important chemotherapeutical agent used to treat epithelial malignancies. High concentrations (20-100 lM) of cisplatin have been used in numerous studies to induce apoptosis of carcinoma cells grown in monolayer culture over 24-48 hr. These conditions may not be relevant to 3-D tumor tissue in vivo and the importance of apoptosis for tumor response is controversial. We here studied the effects of cisplatin on a 3-D colon carcinoma in vitro model (multicellular spheroids). Cisplatin at a dose of 40 lM induced active caspase-3 preferentially in the peripheral 30 lm cell layer of spheroids, mainly during late stages (72-96 hr). The p53 response to cisplatin was also largely confined to peripheral cell layers. Despite the use of a high cisplatin concentration, a significant fraction of the cells in the spheroids survived treatment. A high proportion of surviving cells stained positive for b-galactosidase, a marker of premature senescence. Cells growtharrested by cisplatin treatment showed a higher spontaneous cell death rate than untreated proliferating cells. We propose that acute apoptosis is of minor significance for the overall response of carcinoma cells to cisplatin treatment. ' UICCKey words: cisplatin; apoptosis; senescence; p21Cip1; p27Kip1 Platinum derivatives are important chemotherapeutical agents for many epithelial malignancies, including testicular, ovarian, squamous cell and colon cancers.1,2 In the low intracellular chloride concentrations, cisplatin undergoes equation to form molecules that are reactive with cellular macromolecules. Cisplatin forms covalent bonds to the N7 positions of DNA purines leading to formation intra-or interstrand crosslinks, but also reacts with RNA and protein. 3,4 Treatment of tumor cells in vitro with cisplatin results in apoptosis within 24-48 hr.4-6 Dysregulation of apoptosis pathways is generally assumed to be important for resistance to cisplatin.6 Cisplatin-induced calcium signaling, leading to activation of calpain and cleavage of Bid, 7 and sustained activation of N-terminal-c-Jun kinase, 8 is important for apoptosis signaling. Recent work has shown that cisplatin is able to induce caspase activation in enucleated cells, demonstrating that cisplatin can trigger apoptosis pathways that are independent of its effects on nuclear DNA.9,10 This cytoplasmic pathway involves reactive oxygen species (ROS) and calcium signaling.10 According to one model, ROS induces activation of Bak, leading to VDAC1 and Bax activation. 11Despite the very large number of studies in this area (2,970 articles on MedLine on cisplatin/apoptosis March 2009), it is controversial whether apoptosis is the primary mode of cell death of human carcinoma cells in response to DNA damaging therapeutic drugs, and also whether apoptosis is necessary for the therapeutic effect of such drugs. 10,[12][13][14][15][16] The importance of apoptosis appears to be most pronounced in short-term experiments (1-2 days) and is of lower significance when cell viability is examined after lo...

Massive induction of apoptosis of multicellular tumor spheroids by a novel compound with a calmodulin inhibitor-like mechanism

“…The response of HCT116 MCS to three anticancer agents, cisplatin, melphalan and vinblastine is shown in (Figure 1B). These drugs elicited only limited activation of caspase-3 at the spheroid periphery, consistent with previous reports [24,25].…”

“…Therefore, the ability of cisplatin to reduce clonogenicity of MCS was larger than expected from the limited induction of apoptosis (see Figure 2C below; discussed in [25]). …”

Cancer cell spheroids as a model to evaluate chemotherapy protocols

“…These drug concentrations are in accordance with those used in previous reports. 22,23 The use of such high doses of these drugs is supported on the one hand by the doxorubicin IC 50 above 100 μM in NCI-ADR-RES spheroids, 6 and on the other hand by the data on limited peripheral penetration of cisplatin and doxorubicin in HCT116 spheroids. 23 Induction of cell death by this pretreatment was confirmed by TUNEL staining (Fig.…”

“…5,21 As cells within the spheroid core are exposed to suboptimal drug concentrations, [22][23][24] high concentrations and/or drug combinations are needed to harness antineoplastic agents efficiency. NCI-ADR-RES, doxorubicin-resistant spheroids, were used as a model in our study.…”

Ascitic Fluid in Ovarian Carcinoma – From Pathophysiology to the Treatment

“…Spheroids have low levels of E-cadherin, express epithelial cell adhesion molecule (EpCAM) and cytokeratin, and have a pronounced ability of invasion and recurrence and a more rapid occurrence of ascites. This type of malignant cells with the aggregates is chemoresistant due to limited drug penetration through these multicellular cell aggregates [79,80]. These spheroid cells mimic traits of cancer stem cells (CSC).…”