Understanding cisplatin resistance using cellular models

Many mechanisms of cisplatin resistance have been proposed from studies of cellular models of resistance including changes in cellular drug accumulation, detoxification of the drug, inhibition of apoptosis and repair of the DNA adducts. A series of resistant models were developed from CCRF-CEM leukaemia cells with increasing doses of cisplatin from 100 ng/ml. This produced increasing resistance up to 7-fold with a treatment dose of 1.6 μg/ml. Cisplatin resistance in these cells correlated with increases in the antioxidant glutathione, yet treatment with buthionine sulphoximine, an inhibitor of glutathione synthesis, had no effect on resistance, suggesting that the increase in glutathione was not directly involved in cisplatin resistance. Two models were developed from H69 SCLC cells, H69-CP and H69CIS200 using 100 ng/ml or 200 ng/ml cisplatin respectively. Both cell models were 2-4 fold resistant to cisplatin, and have decreased expression of p21 which may increase the cell’s ability to progress through the cell cycle in the presence of DNA damage. Both the H69-CP and H69CIS200 cells showed no decrease in cellular cisplatin accumulation. However, the H69-CP cells have increased levels of cellular glutathione and are cross resistant to radiation whereas the H69CIS200 cells have neither of these changes. This suggests that increases in glutathione may contribute to cross-resistance to other drugs and radiation, but not directly to cisplatin resistance. There are multiple resistance mechanisms induced by cisplatin treatment, even in the same cell type. How then should cisplatin-resistant cancers be treated? Cisplatin-resistant cell lines are often more sensitive to another chemotherapeutic drug paclitaxel (H69CIS200), or are able to be sensitised to cisplatin with paclitaxel pre-treatment (H69-CP). The understanding of this sensitisation by paclitaxel using cell models of cisplatin resistance will lead to improvements in the clinical treatment of cisplatin resistant tumours

Assessment of low-dose cisplatin as a model of nausea and emesis in beagle dogs, potential for repeated administration

Cisplatin is a highly emetogenic cancer chemotherapy agent, which is often used to induce nausea and emesis in animal models. The cytotoxic properties of cisplatin also cause adverse events that negatively impact on animal welfare preventing repeated administration of cisplatin. In this study, we assessed whether a low (subclinical) dose of cisplatin could be utilized as a model of nausea and emesis in the dog while decreasing the severity of adverse events to allow repeated administration. The emetic, nausea-like behavior and potential biomarker response to both the clinical dose (70 mg/m2) and low dose (15 mg/m2) of cisplatin was assessed. Plasma creatinine concentrations and granulocyte counts were used to assess adverse effects on the kidneys and bone marrow, respectively. Nausea-like behavior and emesis was induced by both doses of cisplatin, but the latency to onset was greater in the low-dose group. No significant change in plasma creatinine was detected for either dose groups. Granulocytes were significantly reduced compared with baseline (P = 0.000) following the clinical, but not the low-dose cisplatin group. Tolerability of repeated administration was assessed with 4 administrations of an 18 mg/m2 dose cisplatin. Plasma creatinine did not change significantly. Cumulative effects on the granulocytes occurred, they were significantly decreased (P = 0.03) from baseline at 3 weeks following cisplatin for the 4th administration only. Our results suggest that subclinical doses (15 and 18 mg/m2) of cisplatin induce nausea-like behavior and emesis but have reduced adverse effects compared with the clinical dose allowing for repeated administration in crossover studies

European mtDNA Variants Are Associated With Differential Responses to Cisplatin, an Anticancer Drug: Implications for Drug Resistance and Side Effects.

Background: Cisplatin, a powerful antitumor agent, causes formation of DNA adducts, and activation of apoptotic pathways. Presently, cisplatin resistance develops in up to 70% of patients but the underlying molecular mechanism(s) are unclear and there are no markers to determine which patients will become resistant. Mitochondria play a significant role not only in energy metabolism but also retrograde signaling (mitochondria to nucleus) that modulates inflammation, complement, and apoptosis pathways. Maternally inherited mitochondrial (mt) DNA can be classified into haplogroups representing different ethnic populations that have diverse susceptibilities to diseases and medications. Methods: Transmitochondrial cybrids, where all cell lines possess identical nuclear genomes but either the H (Southern European) or J (Northern European) mtDNA haplogroups, were treated with cisplatin and analyzed for differential responses related to viability, oxidative stress, and expression levels of genes associated with cancer, cisplatin-induced nephrotoxicity and resistance, apoptosis and signaling pathways. Results: The cisplatin-treated-J cybrids showed greater loss of cell viability along with lower levels of reactive oxygen species and mitochondrial membrane potential compared to cisplatin-treated-H cybrids. After cisplatin treatment, J cybrids showed increased gene expression of BAX, CASP3, and CYP51A, but lower levels of SFRP1 compared to untreated-J cybrids. The cisplatin-treated-H cybrids had elevated expression of CDKN1A/P21, which has a role in cisplatin toxicity, compared to untreated-H cybrids. The cisplatin-treated H had higher transcription levels of ABCC1, DHRS2/HEP27, and EFEMP1 compared to cisplatin-treated-J cybrids. Conclusions: Cybrid cell lines that contain identical nuclei but either H mtDNA mitochondria or J mtDNA mitochondria respond differently to cisplatin treatments suggesting involvement of the retrograde signaling (from mitochondria to nucleus) in the drug-induced cell death. Varying toxicities and transcription levels of the H vs. J cybrids after cisplatin treatment support the hypothesis that mtDNA variants play a role in the expression of genes affecting resistance and side effects of cisplatin

Treating cisplatin-resistant cancer: a systematic analysis of oxaliplatin or paclitaxel salvage chemotherapy

Objective: To examine the pre-clinical and clinical evidence for the use of oxaliplatin or paclitaxel salvage chemotherapy in patients with cisplatin-resistant cancer. Methods: Medline was searched for 1) Cell models of acquired resistance reporting cisplatin, oxaliplatin and paclitaxel sensitivities and 2) Clinical trials of single agent oxaliplatin or paclitaxel salvage therapy for cisplatin/carboplatin-resistant ovarian cancer. Results: Oxaliplatin - Oxaliplatin is widely regarded as being active in cisplatin-resistant cancer. In contrast, data in cell models suggests that there is cross-resistance between cisplatin and oxaliplatin in cellular models with resistance levels which reflect clinical resistance (<10 fold). Oxaliplatin as a single agent had a poor response rate in patients with cisplatin-resistant ovarian cancer (8%, n=91). Oxaliplatin performed better in combination with other agents for the treatment of platinum-resistant cancer suggesting that the benefit of oxaliplatin may lie in its more favourable toxicity and ability to be combined with other drugs rather than an underlying activity in cisplatin resistance. Oxaliplatin therefore should not be considered broadly active in cisplatin-resistant cancer. Paclitaxel – Cellular data suggests that paclitaxel is active in cisplatin-resistant cancer. 68.1% of cisplatin-resistant cells were sensitive to paclitaxel. Paclitaxel as a single agent had a response rate of 22% in patients with platinum-resistant ovarian cancer (n = 1918), a significant increase from the response of oxaliplatin (p<0.01). Paclitaxel-resistant cells were also sensitive to cisplatin, suggesting that alternating between agents may be beneficial. Studies of single agent paclitaxel in platinum-resistant ovarian cancer where patients had previously received paclitaxel had an improved response rate of 35.3% n=232 (p<0.01), suggesting that pre-treatment with paclitaxel improves the response of salvage paclitaxel therapy. Conclusions: Cellular models reflect the resistance observed in the clinic as the cross resistant agent oxaliplatin has a lower response rate compared to the non-cross resistant agent paclitaxel in cisplatin-resistant ovarian cancer. Alternating therapy with cisplatin and paclitaxel may therefore lead to an improved response rate in ovarian cancer

Oxaliplatin induces drug resistance more rapidly than cisplatin in H69 small cell lung cancer cells

Cisplatin produces good responses in solid tumours including small cell lung cancer (SCLC) but this is limited by the development of resistance. Oxaliplatin is reported to show activity against some cisplatin-resistant cancers but there is little known about oxaliplatin in SCLC and there are no reports of oxaliplatin resistant SCLC cell lines. Studies of drug resistance mainly focus on the cellular resistance mechanisms rather than how the cells develop resistance. This study examines the development of cisplatin and oxaliplatin resistance in H69 human SCLC cells in response to repeated treatment with clinically relevant doses of cisplatin or oxaliplatin for either 4 days or 2h. Treatments with 200ng/ml cisplatin or 400ng/ml oxaliplatin for 4 days produced sublines (H69CIS200 and H69OX400 respectively) that showed low level (approximately 2-fold) resistance after 8 treatments. Treatments with 1000ng/ml cisplatin or 2000ng/ml oxaliplatin for 2h also produced sublines, however these were not stably resistant suggesting shorter treatment pulses of drug may be more effective. Cells survived the first five treatments without any increase in resistance, by arresting their growth for a period and then regrowing. The period of growth arrest was reduced after the sixth treatment and the H69CIS200 and H69OX400 sublines showed a reduced growth arrest in response to cisplatin and oxaliplatin treatment suggesting that "regrowth resistance" initially protected against drug treatment and this was further upregulated and became part of the resistance phenotype of these sublines. Oxaliplatin dose escalation produced more surviving sublines than cisplatin dose escalation but neither set of sublines were associated with increased resistance as determined by 5-day cytotoxicity assays, also suggesting the involvement of regrowth resistance. The resistant sublines showed no change in platinum accumulation or glutathione levels even though the H69OX400 subline was more sensitive to buthionine sulfoximine treatment. The H69CIS200 cells were cross-resistant to oxaliplatin demonstrating that oxaliplatin does not have activity against low level cisplatin resistance. Relative to the H69 cells, the H69CIS200 and H69OX400 sublines were more sensitive to paclitaxel and taxotere suggests the taxanes may be useful in the treatment of platinum resistant SCLC. These novel cellular models of cisplatin and oxaliplatin resistant SCLC will be useful in developing strategies to treat platinum-resistant SCLC

Epigallocatechin-3-gallate, a prototypic chemopreventative agent for protection against cisplatin-based ototoxicity

AbstractCisplatin-induced ototoxicity is one of the major factors limiting cisplatin chemotherapy. Ototoxicity results from damage to outer hair cells (OHCs) and other regions of the cochlea. At the cellular level, cisplatin increases reactive oxygen species (ROS) leading to cochlear inflammation and apoptosis. Thus, ideal otoprotective drugs should target oxidative stress and inflammatory mechanisms without interfering with cisplatin's chemotherapeutic efficacy. In this study, we show that epigallocatechin-3-gallate (EGCG) is a prototypic agent exhibiting these properties of an effect otoprotective agent. Rats administered oral EGCG demonstrate reduced cisplatin-induced hearing loss, reduced loss of OHCs in the basal region of the cochlea and reduced oxidative stress and apoptotic markers. EGCG also protected against the loss of ribbon synapses associated with inner hair cells and Na+/K+ ATPase α1 in the stria vascularis and spiral ligament. In vitro studies showed that EGCG reduced cisplatin-induced ROS generation and ERK1/2 and signal transducer and activator of transcription-1 (STAT1) activity, but preserved the activity of STAT3 and Bcl-xL. The increase in STAT3/STAT1 ratio appears critical for mediating its otoprotection. EGCG did not alter cisplatin-induced apoptosis of human-derived cancer cells or cisplatin antitumor efficacy in a xenograft tumor model in mice because of its inability to rescue the downregulation of STAT3 in these cells. These data suggest that EGCG is an ideal otoprotective agent for treating cisplatin-induced hearing loss without compromising its antitumor efficacy.</jats:p

The endocannabinoid/cannabinoid receptor 2 system protects against cisplatin-induced hearing loss

Previous studies have demonstrated the presence of cannabinoid 2 receptor (CB2R) in the rat cochlea which was induced by cisplatin. In an organ of Corti-derived cell culture model, it was also shown that an agonist of the CB2R protected these cells against cisplatin-induced apoptosis. In the current study, we determined the distribution of CB2R in the mouse and rat cochleae and examined whether these receptors provide protection against cisplatin-induced hearing loss. In a knock-in mouse model expressing the CB2R tagged with green fluorescent protein, we show distribution of CB2R in the organ of Corti, stria vascularis, spiral ligament and spiral ganglion cells. A similar distribution of CB2R was observed in the rat cochlea using a polyclonal antibody against CB2R. Trans-tympanic administration of (2-methyl-1-propyl-1H-indol-3-yl)-1-naphthalenylmethanone (JWH015), a selective agonist of the CB2R, protected against cisplatin-induced hearing loss which was reversed by blockade of this receptor with 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630), an antagonist of CB2R. JWH015 also reduced the loss of outer hair cells (OHCs) in the organ of Corti, loss of inner hair cell (IHC) ribbon synapses and loss of Na+/K+-ATPase immunoreactivity in the stria vascularis. Administration of AM630 alone produced significant hearing loss (measured by auditory brainstem responses) which was not associated with loss of OHCs, but led to reductions in the levels of IHC ribbon synapses and strial Na+/K+-ATPase immunoreactivity. Furthermore, knock-down of CB2R by trans-tympanic administration of siRNA sensitized the cochlea to cisplatin-induced hearing loss at the low and middle frequencies. Hearing loss induced by cisplatin and AM630 in the rat was associated with increased expression of genes for oxidative stress and inflammatory proteins in the rat cochlea. In vitro studies indicate that JWH015 did not alter cisplatin-induced killing of cancer cells suggesting this agent could be safely used during cisplatin chemotherapy. These data unmask a protective role of the cochlear endocannabinoid/CB2R system which appears tonically active under normal conditions to preserve normal hearing. However, an exogenous agonist is needed to boost the activity of endocannabinoid/CB2R system for protection against a more traumatic cochlear insult, as observed with cisplatin administration.</p

Epigenetic and antitumor effects of platinum(IV)-octanoato conjugates

We present the anticancer properties of cis, cis, trans-[Pt(IV)(NH3)2Cl2(OA)2] [Pt(IV)diOA] (OA = octanoato), Pt(IV) derivative of cisplatin containing two OA units appended to the axial positions of a six-coordinate Pt(IV) center. Our results demonstrate that Pt(IV)diOA is a potent cytotoxic agent against many cancer cell lines (the IC50 values are approximately two orders of magnitude lower than those of clinically used cisplatin or Pt(IV) derivatives with biologically inactive axial ligands). Importantly, Pt(IV)diOA overcomes resistance to cisplatin, is significantly more potent than its branched Pt(IV) valproato isomer and exhibits promising in vivo antitumor activity. The potency of Pt(IV)diOA is a consequence of several factors including enhanced cellular accumulation correlating with enhanced DNA platination and cytotoxicity. Pt(IV)diOA induces DNA hypermethylation and reduces mitochondrial membrane potential in cancer cells at levels markedly lower than the IC50 value of free OA suggesting the synergistic action of platinum and OA moieties. Collectively, the remarkable antitumor effects of Pt(IV)diOA are a consequence of the enhanced cellular uptake which makes it possible to simultaneously accumulate high levels of both cisplatin and OA in cells. The simultaneous dual action of cisplatin and OA by different mechanisms in tumor cells may result in a markedly enhanced and unique antitumor effects of Pt(IV) prodrugs

New trends for metal complexes with anticancer activity

Medicinal inorganic chemistry can exploit the unique properties of metal ions for the design of new drugs. This has, for instance, led to the clinical application of chemotherapeutic agents for cancer treatment, such as cisplatin. The use of cisplatin is, however, severely limited by its toxic side-effects. This has spurred chemists to employ different strategies in the development of new metal-based anticancer agents with different mechanisms of action. Recent trends in the field are discussed in this review. These include the more selective delivery and/or activation of cisplatin-related prodrugs and the discovery of new non-covalent interactions with the classical target, DNA. The use of the metal as scaffold rather than reactive centre and the departure from the cisplatin paradigm of activity towards a more targeted, cancer cell-specific approach, a major trend, are discussed as well. All this, together with the observation that some of the new drugs are organometallic complexes, illustrates that exciting times lie ahead for those interested in ‘metals in medicine

Small inhibitor of Bcl-2, HA14-1, selectively enhanced the apoptotic effect of cisplatin by modulating Bcl-2 family members in MDA-MB-231 breast cancer cells

Inhibition or downregulation of Bcl-2 represents a new therapeutic approach to by-pass chemoresistance in cancer cells. Therefore, we explored the potential of this approach in breast cancer cells. Cisplatin and paclitaxel induced apoptosis in a dose-dependent manner in MCF-7 (drug-sensitive) and MDA-MB-231 (drug-insensitive) cells. Furthermore, when we transiently silenced Bcl-2, both cisplatin and paclitaxel induced apoptosis more than parental cells. Dose dependent induction of apoptosis by drugs was enhanced by the pre-treatment of these cells with HA14-1, a Bcl-2 inhibitor. Although the effect of cisplatin was significant on both cell lines, the effect of paclitaxel was much less potent only in MDA-MB-231 cells. To further understand the distinct role of drugs in MDA-MB-231 cells pretreated with HA14-1, caspases and Bcl-2 family proteins were studied. The apoptotic effect of cisplatin with or without HA14-1 pre-treatment is shown to be caspase-dependent. Among pro-apoptotic Bcl-2 proteins, Bax and Puma were found to be up-regulated whereas Bcl-2 and Bcl-x(L) were down-regulated when cells were pretreated with HA14-1 followed by paclitaxel or cisplatin. Enforced Bcl-2 expression in MDA-MB-231 cells abrogated the sensitizing effect of HA14-1 in cisplatin induced apoptosis. These results suggest that the potentiating effect of HA14-1 is drug and cell type specific and may not only depend on the inhibition of Bcl-2. Importantly, alteration of other pro-apoptotic or anti-apoptotic Bcl-2 family members may dictate the apoptotic response when HA14-1 is combined with chemotherapeutic drugs