Imaging the pharmacodynamics of HER2 degradation in response to Hsp90 inhibitors

The development of therapeutic inhibitors of key signaling pathways has been hampered by the inability to assess the effect of a drug on its target in the patient. 17-allylaminogeldanamycin (17-AAG) is the first Hsp90 inhibitor to be tested in a clinical trial. It causes the degradation of HER2 and other Hsp90 targets, and has antitumor activity in preclinical models. We have developed a method for imaging the inhibition of Hsp90 by 17-AAG. We labeled an F(ab')2 fragment of the anti-HER2 antibody Herceptin with 68Ga, a positron emitter, which allows the sequential positron-emission tomographic imaging of HER2 expression. We have used this method to quantify as a function of time the loss and recovery of HER2 induced by 17-AAG in animal tumors. This approach allows noninvasive imaging of the pharmacodynamics of a targeted drug and will facilitate the rational design of combination therapy based on target inhibition.     

Evaluation of the Antitumor Activity of Dacomitinib in Models of Human Bladder Cancer

Members of the human epidermal growth factor receptor (HER) family play a significant role in bladder cancer progression and may underlie the development of chemotherapy resistance. Dacomitinib is an irreversible tyrosine kinase inhibitor with structural specificity for the catalytic domains of epidermal growth factor receptor (EGFR), HER2 and HER4 that has exhibited vigorous efficacy against other solid tumors. We evaluated the antitumor activity of dacomitinib in human bladder cancer cell lines expressing varying levels of HER family receptors. These cell lines also were established as bladder cancer xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to assess dacomitinib activity in vivo. Significant cytotoxic and cytostatic effects were noted in cells expressing elevated levels of the dacomitinib target receptors with apoptosis and cell cycle arrest being the predominant mechanisms of antitumor activity. Cells expressing lower levels of HER receptors were much less sensitive to dacomitinib. Interestingly, dacomitinib was more active than either trastuzumab or cetuximab in vitro, and exhibited increased growth inhibition of bladder tumor xenografts compared with lapatinib. Pharmacodynamic effects of dacomitinib included decreased E-cadherin (E-cad) expression, reduction of EGFR and extracellular signal-regulated kinase (ERK) phosphorylation and reduced mitotic count. Dacomitinib also inhibited tumor growth in a chemotherapy-resistant xenograft and, when combined with chemotherapy in a sensitive xenograft, exhibited superior antitumor effects compared with individual treatments. Evaluation in xenograft-bearing mice revealed that this combination was broadly feasible and well tolerated. In conclusion, dacomitinib exhibited pronounced activity both as a single agent and when combined with chemotherapy in human bladder cancer models. Further investigation of dacomitinib in the preclinical and clinical trial settings is being pursued.     

Intratumoral injection of therapeutic HPV vaccinia vaccine following cisplatin enhances HPV-specific antitumor effects

Despite the conventional treatments of radiation therapy and chemotherapy, the 5-year survival rates for patients with advanced-stage cervical cancers remain low. Cancer immunotherapy has emerged as an alternative, innovative therapy that may improve survival. Here, we utilize a preclinical HPV-16 E6/E7-expressing tumor model, TC-1, and employ the chemotherapeutic agent cisplatin to generate an accumulation of CD11c+ dendritic cells in tumor loci making it an ideal location for the administration of therapeutic vaccines. Following cisplatin treatment, we tested different routes of administration of a therapeutic HPV vaccinia vaccine encoding HPV-16 E7 antigen (CRT/E7-VV). We found that TC-1 tumor-bearing C57BL/6 mice treated with cisplatin and intratumoral injection of CRT/E7-VV significantly increased E7-specific CD8+ T cells in the blood and generated potent local and systemic antitumor immune responses compared to mice receiving cisplatin and CRT/E7-VV intraperitoneally or mice treated with cisplatin alone. We further extended our study using a clinical grade recombinant vaccinia vaccine encoding HPV-16/18 E6/E7 antigens (TA-HPV). We found that intratumoral injection with TA-HPV following cisplatin treatment also led to increased E7-specific CD8+ T cells in the blood as well as significantly decreased tumor size compared to intratumoral injection with wild type vaccinia virus. Our study has strong implications for future clinical translation using intratumoral injection of TA-HPV in conjunction with the current treatment strategies for patients with advanced cervical cancer.     

Synergy between chemotherapeutic agents and CTLA-4 blockade in preclinical tumor models

Ipilimumab, a cytotoxic T-lymphocyte antigen-4 (CTLA-4) binding agent, has proven to be an effective monotherapy for metastatic melanoma and has shown antitumor activity in trials when administered with other therapeutic agents. We hypothesized that the combination of ipilimumab with chemotherapeutic agents, such as ixabepilone, paclitaxel, etoposide, and gemcitabine, may produce therapeutic synergy based on distinct but complementary mechanisms of action for each drug and unique cellular targets. This concept was investigated using a mouse homolog of ipilimumab in preclinical murine tumor models, including SA1N fibrosarcoma, EMT-6 mammary carcinoma, M109 lung carcinoma, and CT-26 colon carcinoma. Results of CTLA-4 blockade in combination with one of various chemotherapeutic agents demonstrate that synergy occurs in settings where either agent alone was not effective in inducing tumor regression. Furthermore, when combined with CTLA-4 blockade, ixabepilone, etoposide, and gemcitabine elicited prolonged antitumor effects in some murine models with induction of a memory immune response. Future investigations are warranted to determine which specific chemo-immunotherapy combinations, if any, will produce synergistic antitumor effects in the clinical setting.     

HER2 phosphorylation is maintained by a PKB negative feedback loop in response to anti-HER2 herceptin in breast cancer

Herceptin (trastuzumab) is used in patients with breast cancer who have HER2 (ErbB2)–positive tumours. However, its mechanisms of action and how acquired resistance to Herceptin occurs are still poorly understood. It was previously thought that the anti-HER2 monoclonal antibody Herceptin inhibits HER2 signalling, but recent studies have shown that Herceptin does not decrease HER2 phosphorylation. Its failure to abolish HER2 phosphorylation may be a key to why acquired resistance inevitably occurs for all responders if Herceptin is given as monotherapy. To date, no studies have explained why Herceptin does not abolish HER2 phosphorylation. The objective of this study was to investigate why Herceptin did not decrease HER2 phosphorylation despite being an anti-HER2 monoclonal antibody. We also investigated the effects of acute and chronic Herceptin treatment on HER3 and PKB phosphorylation in HER2-positive breast cancer cells. Using both Förster resonance energy transfer (FRET) methodology and conventional Western blot, we have found the molecular mechanisms whereby Herceptin fails to abolish HER2 phosphorylation. HER2 phosphorylation is maintained by ligand-mediated activation of EGFR, HER3, and HER4 receptors, resulting in their dimerisation with HER2. The release of HER ligands was mediated by ADAM17 through a PKB negative feedback loop. The feedback loop was activated because of the inhibition of PKB by Herceptin treatment since up-regulation of HER ligands and ADAM17 also occurred when PKB phosphorylation was inhibited by a PKB inhibitor (Akt inhibitor VIII, Akti-1/2). The combination of Herceptin with ADAM17 inhibitors or the panHER inhibitor JNJ-26483327 was able to abrogate the feedback loop and decrease HER2 phosphorylation. Furthermore, the combination of Herceptin with JNJ-26483327 was synergistic in tumour inhibition in a BT474 xenograft model. We have determined that a PKB negative feedback loop links ADAM17 and HER ligands in maintaining HER2 phosphorylation during Herceptin treatment. The activation of other HER receptors via ADAM17 may mediate acquired resistance to Herceptin in HER2-overexpressing breast cancer. This finding offers treatment opportunities for overcoming resistance in these patients. We propose that Herceptin should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for patients with HER2-positive breast cancer. Our results may also have implications for resistance to other therapies targeting HER receptors.     

Antineoplastic effect of mer-trichlorobisdimethylsulphoxideaminorutheniumIII against murine tumors: comparison with cisplatin and with ImH[RuIm2Cl4

An asymmetric rutheniumIII complex containing dimethylsulphoxide ligands, namely mer-trichlorobisdimethylsulphoxideaminorutheniumIII (BBR2382), has been tested in mice bearing solid metastasizing tumors. The effects of i.p. treatment with BBR2382 on primary tumor growth and on the survival time of hosts carrying s.c. or i.m. tumors have been compared to those of cisplatin and of a rutheniumIII complex with imidazole ligands, ImH[RuIm2Cl4], described as a potent antitumor agent in a number of experimental models of murine neoplasms. In mice bearing Lewis lung carcinoma, BBR2382 results as effective as cisplatin on s.c. primary tumor growth and more potent than cisplatin on the prolongation of host survival time. The combined treatment of mice bearing Lewis lung carcinoma with cisplatin and BBR2382 causes a reduction of s.c. tumors higher than that caused by each single agent; the effects on host survival time are similar to those caused by BBR2382 alone but significantly superior to those caused by cisplatin alone. In CBA mice bearing MCa mammary carcinoma, the effects of BBR2382 are slightly lower than those of cisplatin on i.m. tumors but are equivalent on host survival time. The comparison of the antineoplastic action of BBR2382 with that of ImH[RuIm2Cl4] is always in favor of the former, independently of the parameter chosen and of the tumor system used. Qualitatively, the antitumor action of BBR2382 seems different from that of cisplatin and of ImH[RuIm2Cl4]; it is supposed that this agent, like other "rutheniumIII dimethylsulphoxide" complexes, could have a particular efficacy for tumors localized in the lungs.     

Effect of cisplatin upon expression of in vivo immune tumor resistance

The major intent of cancer treatment with cytotoxic drugs is direct tumor cell damage, but some of these drugs have been shown to be immunomodulatory. Cisplatin is a widely used cytotoxic drug that has been combined with biological response modifiers in recent clinical trials. To evaluate further whether cisplatin may independently alter the level of host resistance against tumor growth, the drug was tested in the Mc7 sarcoma rat tumor model. The expression of in vivo tumor resistance against Mc7 sarcoma in syngeneic Wistar rats is mediated by circulating non-cytotoxic T lymphocytes. These cells interact specifically with tumor cells to generate cytotoxic effectors locally at the site of a tumor challenge. Activities of these components of expression of tumor resistance were measured in vivo after administration of cisplatin and dose-dependent effects were found. Low-dose cisplatin (0.3 mg/kg) increased the activity of the circulating lymphocytes that mediate tumor resistance, and high-dose cisplatin (9 mg/kg) suppressed both mediator lymphocyte activity and the generation of antitumor effector mechanisms. These studies suggest that low-dose cisplatin may be immunomodulatory and combining it with biological response modifiers might be a useful strategy. However, high-dose cisplatin given with biological response modifiers may negate potential immunomodulatory activities of such agents.     

High dose cisplatin compared with high dose cyclophosphamide in the management of advanced epithelial ovarian cancer (FIGO stages III and IV): report from the North Thames Cooperative Group.

A randomised study comparing cisplatin 120 mg intravenously with cyclophosphamide 2 g intravenously, each drug being given every month for six months followed by a low dose regimen for a further six months in responding patients, was carried out in 86 patients with advanced epithelial ovarian carcinoma (FIGO stages III and IV). Patients given cisplatin were found to have a longer median survival time than those given cyclophosphamide (19 months compared with 12 months) and a longer median duration of complete clinical response (18 months compared with eight months). The difference in disease free survival was statistically significant even after factors such as age, stage of disease, and the completeness of initial surgery had been taken into account. This study suggests that cisplatin is a more effective chemotherapeutic agent than cyclophosphamide for advanced ovarian cancer and should be the agent of choice in future trials comparing combination chemotherapy with a single agent.     

Preclinical Combination Therapy of Thiarabine Plus Various Clinical Anticancer Agents

Thiarabine is undergoing clinical trials. In support of that effort combination therapy of thiarabine plus six clinical anticancer agents was evaluated using various human tumor xenograft models. The antitumor activity of thiarabine in combination appeared to be greater than additive with irinotecan (DLD-1 colon), paclitaxel (PC-3 prostate), cisplatin (PC-3 prostate), or cyclophosphamide (RL lymphoma), additive with irinotecan (NCI-H460 NSCLC), cisplatin (NCI-H460 NSCLC) or methotrexate (CCRF-CEM leukemia), and less than additive with irinotecan (HT29 colon), paclitaxel (NCI-H460 NSCLC) or cisplatin (NCI-H23 NSCLC). Combining thiarabine with irinotecan, paclitaxel, cisplatin, or cyclophosphamide should receive consideration in the clinical treatment of cancer.     

ADP sensitizes ZL55 cells to the activity of cisplatin

Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor in which cisplatin therapy is commonly used, although its effectiveness is limited. It follows that research efforts dedicated to identify promising combinations that can synergistically kill cancer cells are needed. Because we recently demonstrated that ADP inhibits the proliferation of ZL55 cells, an MPM-derived cell line obtained from bioptic samples of asbestos-exposed patients. Our objective in this study was to investigate the hypothesis that ADP also potentiates the cytotoxic activity of cisplatin. Results show that in ZL55 cells ADP enhanced (a) the cytotoxicity of cisplatin by 12-fold, (b) the restraint of cell clonogenic potential cisplatin-mediated, and (c) the number of apoptotic cells. Cisplatin, but not ADP, caused caspases activation; nevertheless, poly(ADP-ribose) polymerase-1 was not only cleaved in cisplatin-treated cells but also in cells treated with ADP alone. Furthermore, ADP, but not cisplatin, decreased mTOR and 6SK phosphorylations. Both ADP and cisplatin increased p53 protein, but ADP was also able to enhance p53 messenger RNA. P53 silencing resulted in a very large decrement of cell death induced by ADP or by cisplatin and reverted ADP effects on mTOR/S6K phosphorylation, suggesting that activated p53 may act as a negative regulator of mTOR. Consistently, the inhibition of mTOR by rapamycin also sensitized cells to cisplatin, and the effects of cisplatin plus rapamycin were identical to those obtained with cisplatin plus ADP. These findings suggest that the combination of ADP and cisplatin may be a promising strategy for the clinical treatment of cisplatin-resistant MPM.     

Effective antibody therapy induces host-protective antitumor immunity that is augmented by TLR4 agonist treatment

Toll-like receptors are potent activators of the innate immune system and generate signals leading to the initiation of the adaptive immune response that can be utilized for therapeutic purposes. We tested the hypothesis that combined treatment with a Toll-like receptor agonist and an antitumor monoclonal antibody is effective and induces host-protective antitumor immunity. C57BL/6 human mutated HER2 (hmHER2) transgenic mice that constitutively express kinase-deficient human HER2 under control of the CMV promoter were established. These mice demonstrate immunological tolerance to D5-HER2, a syngeneic human HER2-expressing melanoma cell line. This human HER2-tolerant model offers the potential to serve as a preclinical model to test both antibody therapy and the immunization potential of human HER2-targeted therapeutics. Here, we show that E6020, a Toll-like receptor-4 (TLR4) agonist effectively boosted the antitumor efficacy of the monoclonal antibody trastuzumab in immunodeficient C57BL/6 SCID mice as well as in C57BL/6 hmHER2 transgenic mice. E6020 and trastuzumab co-treatment resulted in significantly greater inhibition of tumor growth than was observed with either agent individually. Furthermore, mice treated with the combination of trastuzumab and the TLR4 agonist were protected against rechallenge with human HER2-transfected tumor cells in hmHER2 transgenic mouse strains. These findings suggest that combined treatment with trastuzumab and a TLR4 agonist not only promotes direct antitumor effects but also induces a host-protective human HER2-directed adaptive immune response, indicative of a memory response. These data provide an immunological rationale for testing TLR4 agonists in combination with antibody therapy in patients with cancer.     

Study of inhibition effect of Herceptin on interaction between Heregulin and ErbB Receptors HER3/HER2 by single-molecule force spectroscopy

Herceptin is a monoclonal antibody against HER2, which is a member of the epidermal growth factor receptor (ErbB) family and is overexpressed in many cancers. In this work, we have applied single-molecule force spectroscopy to study the effect of Herceptin on HER2 modulated ligand–receptor interaction for ErbB signaling in living cells. Heregulin β1 (HRG), the specific ligand of HER3, was used for HER2 activation as HER3 is the preferable dimerization partner of HER2 and HER3/HER2 is the most representative heterodimer found in cancer. Our results demonstrated a more stable binding of HRG to the cells co-expressing HER3 and HER2 than those expressing HER3 alone. Moreover, the binding force of Herceptin and HER2 is as strong as that of HRG and HER3/HER2. With the addition of Herceptin, the binding strength of HRG to the cells co-expressing HER3 and HER2 decreased. The presence of Herceptin changed the dynamic force spectrum of HRG-HER3/HER2 to that similar to HRG-HER3. Therefore, the enhancement in HRG-HER3 binding after recruiting HER2 was inhibited by Herceptin. The method offers a new approach to study the molecular mechanism of Herceptin anti-cancer effect.     

Rationally designed anti-HER2/neu peptide mimetic disables P185HER2/neu tyrosine kinases in vitro and in vivo

Monoclonal antibodies specific for the p185HER2/neu growth factor receptor represent a significant advance in receptor-based therapy for p185HER2/neu-expressing human cancers. We have used a structure-based approach to develop a small (1.5 kDa) exocyclic anti-HER2/neu peptide mimic (AHNP) functionally similar to an anti-p185HER2/neu monoclonal antibody, 4D5 (Herceptin). The AHNP mimetic specifically binds to p185HER2/neu with high affinity (KD=300 nM). This results in inhibition of proliferation of p185HER2/neu-overexpressing tumor cells, and inhibition of colony formation in vitro and growth of p185HER2/neu-expressing tumors in athymic mice. In addition, the mimetic sensitizes the tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents. A comparison of the molar quantities of the Herceptin antibody and the AHNP mimetic required for inhibiting cell growth and anchorage-independent growth showed generally similar activities. The structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies.     

Modifications in synthesis strategy improve the yield and efficacy of geldanamycin-herceptin immunoconjugates

Geldanamycin (GA) was modified with N-tert-butyloxycarbonyl-1,3-diaminopropane to introduce a latent primary amine. After deprotection, this primary amine provided a site for introduction of a maleimide group that enabled linkage to proteins. This maleimido derivative of geldanamycin (GMB-APA-GA) was linked to the monoclonal antibody Herceptin after the antibody had been modified with Traut's reagent to introduce thiol groups. By this sequence, a new immunoconjugate (H:APA-GA) was generated that showed greater antiproliferative activity than the previously reported analogous immunoconjugate created with a 1,4-diaminobutane spacer derivative of geldanamycin to form an immunoconjugate, H:ABA-GA. Both immunoconjugates inhibited in vitro the growth of MDA-361/DYT2 cells, a cell line overexpressing the HER2 antigen, while Herceptin alone was ineffective. However, H:APA-GA showed better efficacy than H:ABA-GA (IC(50) = 0.2 vs 0.58 mg/mL and cell doubling time >12 vs 6 days, respectively). Results of the in vivo therapy experiments in a xenograft model were consistent with the in vitro findings. Treatment with Herceptin prolonged the survival of the tumor-bearing mice when compared with the control group, but H:ABA-GA and H:APA-GA were each more efficacious than unmodified Herceptin. However, unlike H:ABA-GA, the immunoconjugate H:APA-GA caused stable tumor regression (in 25% of the recipients), showing a qualitative improvement with potential clinical relevance.     

Effects of electrogenetherapy with p53wt combined with cisplatin on curvival of human tumor cell lines with different p53 status

The aim of our study was to evaluate electrogenetherapy with p53wt alone or combined with cisplatin on two colorectal (HT-29 and LoVo) and two prostatic (PC-3 and Du145) carcinoma cell lines with different p53 status. In addition, the feasibility of electrogenetherapy with p53wt was tested also in vivo on PC-3 prostatic cancer xenografts. Electrogenetherapy with p53wt was dependent on the p53 status of the cell lines used. Electrogenetherapy was the most effective on the PC-3 (p53 null) and Du145 (p53mt) cells, and to the much lesser extent in LoVo cells (p53wt). The exception was the HT-29 cell line with overexpressed mutated p53, where electrogenetherapy with p53wt was the least effective. Sensitivity of the cell lines to cisplatin was independent of the p53 status. Furthermore, the presence of exogenous p53 due to electrogenetherapy did not enhance cisplatin cytotoxicity, since the combination of these therapies resulted in additive cytotoxic effect. The effectiveness of electrogenetherapy with p53wt was also demonstrated in vivo by successful treatment of subcutaneous PC-3 tumors in mice. In conclusion, our study shows that electrogenetherapy with p53wt is feasible, and resulted in comparable cytotoxic and antitumor effectiveness to viral-mediated p53wt gene therapy. This therapy was effective and dependent on the p53 status of the tumor cell lines. Combination of electrogenetherapy and cisplatin resulted in additional cell kill by cisplatin, and was not dependent on the p53 status.     

Preclinical evaluation of the proteasome inhibitor bortezomib in cancer therapy

Bortezomib is a highly selective, reversible inhibitor of the 26S proteasome that is indicated for single-agent use in the treatment of patients with multiple myeloma who have received at least 2 prior therapies and are progressing on their most recent therapy. Clinical investigations have been completed or are under way to evaluate the safety and efficacy of bortezomib alone or in combination with chemotherapy in multiple myeloma, both at relapse and presentation, as well as in other cancer types. The antiproliferative, proapoptotic, antiangiogenic, and antitumor activities of bortezomib result from proteasome inhibition and depend on the altered degradation of a host of regulatory proteins. Exposure to bortezomib has been shown to stabilize p21, p27, and p53, as well as the proapoptotic Bid and Bax proteins, caveolin-1, and inhibitor κB-α, which prevents activation of nuclear factor κB-induced cell survival pathways. Bortezomib also promoted the activation of the proapoptotic c-Jun-NH₂ terminal kinase, as well as the endoplasmic reticulum stress response. The anticancer effects of bortezomib as a single agent have been demonstrated in xenograft models of multiple myeloma, adult T-cell leukemia, lung, breast, prostate, pancreatic, head and neck, and colon cancer, and in melanoma. In these preclinical in vivo studies, bortezomib treatment resulted in decreased tumor growth, angiogenesis, and metastasis, as well as increased survival and tumor apoptosis. In several in vitro and/or in vivo cancer models, bortezomib has also been shown to enhance the antitumor properties of several antineoplastic treatments. Importantly, bortezomib was generally well tolerated and did not appear to produce additive toxicities when combined with other therapies in the dosing regimens used in these preclinical in vivo investigations. These findings provide a rationale for further clinical trials using bortezomib alone or in combination regimens with chemotherapy, radiation therapy, immunotherapy, or novel agents in patients with hematologic malignancies or solid tumors.     

Electrogene therapy with p53 of murine sarcomas alone or combined with electrochemotherapy using cisplatin

The aim of our study was to evaluate feasibility and therapeutic potential of electrogene therapy with p53 alone or combined with electrochemotherapy using cisplatin on two murine sarcomas with different p53 status. Antitumor effectiveness of three consecutive electrogene treatments with p53 was more effective in wild-type LPB tumors than mutated SA-1 tumors, resulting in 21.4% of tumor cures in LPB tumors and 12.5% in SA-1 tumors. Pretreatment of tumors with electrogene therapy with p53 enhanced chemosensitivity of both tumor models treated by electrochemotherapy with cisplatin. After only one application of this treatment combination in the LPB tumor model, specific tumor growth delay was prolonged in the combined treatment group compared to electrogene therapy with p53 or electrochemotherapy with cisplatin alone, whereas in SA-1 tumors this treatment combination resulted in 31.6% of cured animals. Results of our study show that electrogene therapy with p53 alone or combined with electrochemotherapy is feasible and effective treatment of tumors. The combination of electrogene therapy and electrochemotherapy after only one application resulted in complete regression of tumors.     

Different recognition of DNA modified by aatitumor cisplatin and its clinically ineffective trans isomer by tumor suppressor protein p53

The p53 gene encodes a nuclear phosphoprotein that is biologically activated in response to genotoxic stresses including treatment with anticancer platinum drugs. The DNA binding activity of p53 protein is crucial for its tumor suppressor function. DNA interactions of active wild-type human p53 protein with DNA fragments and oligodeoxyribonucleotide duplexes modified by antitumor cisplatin and its clinically ineffective trans isomer (transplatin) were investigated by using a gel mobility shift assay. It was found that DNA adducts of cisplatin reduced binding affinity of the consensus DNA sequence to p53, whereas transplatin adducts did not. This result was interpreted to mean that the precise steric fit required for the formation and stability of the tetrameric complex of p53 with the consensus sequence cannot be attained, as a consequence of severe conformational perturbations induced in DNA by cisplatin adducts. The results also demonstrate an increase of the binding affinity of p53 to DNA lacking the consensus sequence and modified by cisplatin but not by transplatin. In addition, only major 1,2-GG intrastrand cross-links of cisplatin are responsible for this enhanced binding affinity of p53. The data base on structures of various DNA adducts of cisplatin and transplatin reveals distinctive structural features of 1,2-intrastrand cross-links of cisplatin, suggesting a unique role for this adduct in the binding of p53 to DNA lacking the consensus sequence. The results support the hypothesis that the mechanism of antitumor activity of cisplatin may also be associated with its efficiency to affect the binding affinity of platinated DNA to active p53 protein.     

Enhancement of cisplatin-induced apoptosis by saffron in human lung cancer cells

BackgroundCisplatin is a prevalent chemotherapeutic agent, and it has been used extensively to treat lung cancer. However, its clinical efficacy is hampered by its safety profile and dose-limiting toxicity. Saffron is a natural product that has shown significant anticancer effects. The combination treatment of saffron with chemotherapeutic agents has been considered a new strategy.MethodsHerein, saffron extract as a natural anticancer substance was combined with cisplatin to assess their combined efficacy against tumor development in vitro. In A549 and QU-DB cell lines, the combined effect of the saffron extract with cisplatin led to a significant reduction in cell viability as compared to cisplatin alone.ResultsAfter 48 h incubation a considerable reduction in ROS levels in the QU-DB cell line upon treatment with cisplatin in the presence of saffron extract in comparison with cells treated with cisplatin alone. Furthermore, apoptosis increased significantly when in cells treated with cisplatin in combination with saffron extract compared to cisplatin alone.ConclusionOur data establish that the combination of saffron extract as a natural anticancer substance with cisplatin leads to improved cell toxicity of cisplatin as an anticancer agent. Therefore, the saffron extract could be potentially used as an additive to enable a reduction in cisplatin dosages and its side effects.