P-glycoprotein (ABCB1), multidrug resistance protein-1 (ABCC1) and breast cancer resistance protein (ABCG2) belong to the ATP-binding cassette (ABC) superfamily of proteins that play an important physiological role in protection of the body from toxic xenobiotics and endogenous metabolites. Beyond this, these transporters determine the toxicity profile of many drugs, and confer multidrug resistance (MDR) in cancer cells associated with a poor treatment outcome of cancer patients. It has long been hypothesized that inhibition of ABC drug efflux transporters will increase drug accumulation and thereby overcome MDR, but until now no approved inhibitor of these transporters is available in the clinic. In this review we present molecular strategies to overcome this type of drug resistance and discuss for each of these strategies their promising value or indicate underlying reasons for their limited success.
Aim: CD22ΔE12 as an oncogenic driver lesion in aggressive and drug-resistant B-precursor acute lymphoblastic leukemia (BPL) cells. The purpose of the present study was to identify the CD22ΔE12-specific signature transcriptome in human BPL cells and evaluate the clinical potential of a nanoscale formulation of CD22ΔE12-siRNA as an RNAi therapeutic against drug-resistant BPL. CD22ΔE12-siRNA nanoparticles significantly improved the event-free survival (EFS) outcome of NOD/SCID (NS) mice challenged with human BPL xenograft cells.
Methods: Gene expression and translational bioinformatics methods were applied to examine the expression of the CD22ΔE12-specific signature transcriptome in human BPL cells in subsets of BPL patients. Survival analysis for mice challenged with BPL cells and treated with CD22ΔE12 siRNA was performed using standard methods.
Results: Leukemia cells from CD22ΔE12-Tg mice exhibit gene and protein expression profiles consistent with constitutive activation of multiple signaling networks, mimicking the profiles of relapsed BPL patients as well as newly diagnosed high-risk patients with BCR-ABL+/Philadelphia chromosome (Ph)+ BPL as well as Ph-like BPL. A nanoscale formulation of CD22ΔE12-siRNA abrogated the in vivo clonogenicity of the leukemia-initiating leukemic cell fraction in xenograft specimens derived from patients with relapsed BPL and significantly improved the EFS outcome of NS mice challenged with drug-resistant human BPL xenograft cells.
Conclusion: The CD22-RNAi technology is applicable to all BPL patients both high risk and standard risk. That is because CD22ΔE12 is a characteristic feature of drug-resistant leukemic clones that escape chemotherapy and cause relapse in both high risk and low risk subgroups of patients. The technology therefore has the potential (1) for prevention of relapses by selectively killing the clones that are most likely to escape chemotherapy and cause relapse as well (2) for treatment of relapses in BPL. This research project may also lead to innovative salvage regimens against other forms of CD22ΔE12-positive relapsed B-lineage leukemias and lymphomas.
Aim: Resistance to fluoropyrimidine drugs (FPs) is a major cause of mortality in colorectal cancer (CRC). We assessed the potency advantage of the polymeric FP F10 relative to 5-fluorouracil (5-FU) in four human CRC cell lines that differ only in TP53 mutational status to determine how p53 mutations affect drug response and whether F10 is likely to improve outcomes.
Methods: HCT-116 human CRC cells (p53+/+) and three isogenic variants (p53-/-, R248W/+, R248W/-) were assessed for drug response. Resistance factors were derived from cell viability data and used to establish the relative potency advantage for F10. Rescue studies with exogenous uridine/thymidine determined if cytotoxicity resulted from DNA-directed processes.
Results: Significant resistance to 5-FU resulted from p53-loss or from gain-of-function (GOF) mutation (R248W) and was greatest when GOF mutation was coupled with loss of wild-type p53. F10 is much more potent than 5-FU (137-314-fold depending on TP53 mutational status). F10 and 5-FU induce apoptosis by DNA- and RNA-directed mechanisms, respectively, and only F10 shows a modest enhancement in cytotoxicity upon co-treatment with leucovorin.
Conclusion: TP53 mutational status affects inherent sensitivity to FPs, with p53 GOF mutations most deleterious. F10 is much more effective than 5-FU regardless of TP53 mutations and has potential to be effective to CRC that is resistant to 5-FU due, in part, to TP53 mutations.
Aim: Mitomycin C (MMC) is a commonly used as intravesical treatment for superficial bladder cancer. However, its role in combination with ras inhibition has not been investigated. The aim of this study was to explore the role of ras in MMC-induced apoptosis in T24 bladder cancer cells and to determine the efficacy of combination therapy in vitro.
Methods: We measured the effects of various doses of MMC on apoptosis induction as well as on ras, ERK and Ki-67 protein expression by T24 cell line using immunocytochemistry, flow cytometry and Western blotting. We also tested the effect of siRNA on ras employed singly or in combination with MMC.
Results: T24 cells expressed high level of ras protein. MMC treatment increased the level of ras and ERK protein expression after 24 h, and decreased these levels after 72 h. Ras siRNA (100 nmol/L) caused massive apoptosis associated with a marked decrease in ras expression in T24 cells. When combined with low doses of MMC, ras siRNA (50 nmol/L) sensitized T24 cells to apoptosis and decreased their expression of ras. The effect of combined therapy was higher than that of either compound used alone. Expression levels of ERK, a downstream target of ras, declined following combination therapy.
Conclusion: Ras siRNA in combination with low dose MMC is a possible treatment strategy for patients with ras-positive bladder tumors.
Aim: To investigate the effects of cisplatin on the human non-small cell lung carcinoma (NCI-H460) cell line regarding cytotoxicity, genotoxicity, and expression of genes associated with apoptosis (BIRC5) and autophagy (BECN1).
Methods: Cell cultures were treated with cisplatin concentrations (0.16-33.3 μmol/L) for 48 h. Mutagenicity and acute and chronic cytotoxicities were assessed using the MTT, clonogenic, and cytokinesis-block micronucleus assays. Gene expression of BIRC5 and BECN1 was evaluated by reverse transcription-polymerase chain reaction.
Results: Cisplatin IC50 (0.33 μmol/L) increased micronucleus frequency 2.50 times. Cisplatin was also cytotoxic in the 0.6-33.3 μmol/L range, with reduced expression of the BIRC5 gene, suggesting induction of apoptosis. Besides reducing the expression of the BIRC5 gene, 33.3 μmol/L cisplatin increased the expression of the BECN1 gene, suggesting that autophagy can be related to cisplatin resistance.
Conclusion: Cisplatin inhibited NCI-H460 growth, and cisplatin IC50 induced genotoxic damage. When higher cisplatin concentrations are used, the expression of genes associated with apoptosis and autophagy was changed. This results points to a further investigation of the role of autophagy in cisplatin resistance.