Zahid H. Siddik

Induction of p21 by p53 following DNA damage inhibits both Cdk4 and Cdk2 activities.

DNA damage often activates the p53–p21 pathway and causes G1-phase arrest in mammalian cells. Although there is ample evidence that p21 induction by p53 leads to Cdk2 inhibition, it is unclear whether this checkpoint event also leads to Cdk4 inhibition. Diaminocyclohexane(trans-diacetato)(dichloro) platinum(IV) (DAP), a platinum-based coordination complex, is a DNA-damaging agent that is effective against a variety of tumor cells resistant to the parental drug cisplatin. Our previous studies established that treatment of human cancer cells with low effective concentrations of DAP specifically activates the G1-phase checkpoint and simultaneously inhibit Cdk4 and Cdk2 activities. Here we demonstrate that DAP treatment of human cancer cells activates the p53–p21 pathway without activating other known mechanisms that inhibit Cdk4 and Cdk2 activities. The induced p21 binds to both the Cdk4/cyclin D and Cdk2/cyclin E complexes and inhibits both of their kinase activities. Conversely, inhibition of p21 induction by cycloheximide or by p21 gene deletion prevents DAP-induced inhibition of Cdk4 and Cdk2 activities. Attenuated p53 expression and p21 induction also eliminates DAP-induced G1-phase arrest and inhibition of Cdk4 and Cdk2 activities. Together, these findings establish that activation of the p53–p21 pathway is responsible for the DAP-induced G1-phase checkpoint response and provide the first solid evidence that p21 induction by p53 during a DNA damage-induced G1-phase checkpoint response inhibits both Cdk4 and Cdk2 activities.

Epidermal Growth Factor Receptor (EGFR) Ubiquitination as a Mechanism of Acquired Resistance Escaping Treatment by the Anti-EGFR Monoclonal Antibody Cetuximab

Cetuximab is an epidermal growth factor receptor (EGFR)-blocking antibody that has been approved for treatment of patients with metastatic colorectal cancer. In this study, we investigated biochemical changes in signaling pathways of a cetuximab-resistant subline of DiFi colorectal cancer cells (DiFi5) that was developed by exposing the parental sensitive cells to subeffective doses of cetuximab over an extended period of time. Compared with parental DiFi cells that express high levels of EGFR and in which cetuximab induces apoptosis, the cetuximab-resistant DiFi5 cells showed markedly lower protein levels of EGFR, an increased association of EGFR with Cbl, and an increased ubiquitination of EGFR. DiFi5 cells also had a markedly higher level of Src-Y416 phosphorylation both at baseline and on EGF stimulation. Although EGFR levels were low, DiFi5 cells responded to EGF stimulation with robust phosphorylation of EGFR on Y845 and strong phosphorylation of Akt and extracellular signal-regulated kinase, comparable to those of parental cells. Most importantly, inhibition of Src kinase activity with PP2 reversed the resistance of DiFi5 cells to cetuximab-induced apoptosis without affecting the levels of EGFR in the cells. Our results indicate that colorectal cancer cells may develop acquired resistance to cetuximab via altering EGFR levels through promotion of EGFR ubiquitination and degradation and using Src kinase-mediated cell signaling to bypass their dependency on EGFR for cell growth and survival.

The comparative pharmacokinetics of carboplatin and cisplatin in mice and rats

The plasma, urinary and biliary clearances of cisplatin and its non-nephrotoxic analogue, Carboplatin (cis-diammine-1,1-cyclobutane dicarboxylate platinum II, CBDCA, JM8) have been determined in mice and rats following intravenous administration of the compounds. The plasma concentration-time curves were biphasic during the time period studied (0-60 min), with t1/2 alpha of 2-3 min for both platinum complexes and t1/2 beta of 10-15 min for cisplatin and 25-26 min for Carboplatin. The kinetic rate constants, k12 and k21, were similar for both Carboplatin and cisplatin, indicating that there was no appreciable net accumulation of the compounds in the peripheral tissues. Immediately after administration, Carboplatin became reversibly bound to plasma proteins in vivo to the extent of about 20%. Appreciable irreversible binding appeared after the first 60 min and increased steadily, so that by 4 hr only 34% of the compound was present in the plasma as the free drug. In comparison, binding of cisplatin to plasma was exclusively irreversible and, after the first 10 min, free drug disappeared rapidly, such that by 60 min free platinum was not detectable. The plasma clearance of free cisplatin (26.1 ml/min/kg) was significantly greater than that of either Carboplatin (10.3 ml/min/kg) or insulin (10.1 ml/min/kg). The main route of excretion of the two platinum complexes was via the urine, with 80-90% of Carboplatin and 43-48% of cisplatin being excreted within 4 hr. In the rat, the Carboplatin excreted in the urine was predominantly as the unchanged compound. The renal clearance of cisplatin (12.3 ml/min/kg) was significantly greater than that of either Carboplatin (9.3 ml/min/kg) or insulin (9.6 ml/min/kg), suggesting that cisplatin was excreted by an active renal secretory mechanism whilst Carboplatin was eliminated by glomerular filtration alone. Biliary excretion of the two compounds was only 0.4-1.2% of the administered dose in 6 hr, with biliary clearance of cisplatin (0.27 ml/min/kg) being fivefold greater than that of Carboplatin (0.053 ml/min/kg). The results indicate that the major pharmacokinetic differences between Carboplatin and cisplatin relate to their renal handling and their reactivity with macromolecules. These differences may well underline the substantial lack of Carboplatin nephrotoxicity in comparison with cisplatin.

Flameless atomic absorption spectrophotometric determination of platinum in tissues solubilized in hyamine hydroxide

Processing biological samples by solubilization in Hyamine hydroxide (methylbenzethonium hydroxide) as an alternative to wet ashing with concentrated nitric acid for atomic absorption determination of platinum has been explored. The concentrations of platinum in tissues removed from rats 2 h after they had received the antitumor drug Carboplatin (60 mg/kg, iv) were comparable using either of the procedures, indicating the utility of tissue solubilization. Limits of detection were also comparable between the Hyamine hydroxide (0.25 microgram platinum/g) and nitric acid (0.15 microgram/g) procedures. The solubilization method, however, has advantages over wet digestion because of its simplicity and greater safety.

Therapeutic Targeting of ATP7B in Ovarian Carcinoma

Purpose: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. Experimental Design: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Results:ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC50 levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH2-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. Conclusion: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

Plasma free platinum pharmacokinetics in patients treated with high dose carboplatin

Plasma free platinum (less than 50,000 mol. wt) pharmacokinetics have been studied in eight patients treated with high-dose (800-1600 mg/m2) carboplatin as a 1 h infusion with moderate hydration. Following the infusion, levels decayed biphasically with half-lives (means +/- S.D.) of 83 +/- 15 min and 6.1 +/- 2.8 h. The plasma free platinum area under the concentration vs. time curve (AUC) at 1600 mg/m2 in five patients was 23 +/- 2 mg carboplatin/ml.min. Comparison with data at conventional doses (less than or equal to 500 mg/m2) gave no indication of non-linear kinetics. Total body clearance of free platinum was found to correlate with glomerular filtration rate (r = 0.769, P = 0.03), and haematological toxicity, white cell nadir and duration of thrombocytopenia, correlated with plasma free platinum AUC (r = 0.784, P = 0.02 and r = 0.885, P = 0.01, respectively). Persistence of platinum was demonstrated in tissues removed at autopsy from a patient who had received carboplatin 14 days earlier. Highest platinum levels were found in the liver, kidney, skin and small cell lung tumour.

Elevated glutathione levels confer cellular sensitization to cisplatin toxicity by up-regulation of copper transporter hCtr1

Previous studies have demonstrated that treating cultured cells with cisplatin (CDDP) up-regulated the expression of glutathione (GSH) and its de novo rate-limiting enzyme glutamate-cysteine ligase (GCL), which consists of a catalytic (GCLC) and a modifier (GCLM) subunit. It has also been shown that many CDDP-resistant cell lines exhibit high levels of GCLC/GCLM and GSH. Because the GSH system is the major intracellular regulator of redox conditions that serve as an important detoxification cytoprotector, these results have been taken into consideration that elevated levels of GCL/GSH are responsible for the CDDP resistance. In contrast to this context, we demonstrated here that overexpression of GSH by transfection with an expression plasmid containing the GCLC cDNA conferred sensitization to CDDP through up-regulation of human copper transporter (hCtr) 1, which is also a transporter for CDDP. Depleting GSH levels in these transfected cells reversed CDDP sensitivity with concomitant reduction of hCtr1 expression. Although rates of copper transport were also up-regulated in the transfected cells, these cells exhibited biochemical signature of copper deficiency, suggesting that GSH functions as an intracellular copper-chelator and that overexpression of GSH can alter copper metabolism. More importantly, our results reveal a new role of GSH in the regulation of CDDP sensitivity. Overproduction of GSH depletes the bioavailable copper pool, leading to up-regulation of hCtr1 and sensitization of CDDP transport and cell killing. These findings also have important implications in that modulation of the intracellular copper pool may be a novel strategy for improving chemotherapeutic efficacy of platinum-based antitumor agents.

Tissue Platinum Concentration and Tumor Response in Non–Small-Cell Lung Cancer

PURPOSE Platinum resistance is a major limitation in the treatment of advanced non-small-cell lung cancer (NSCLC). Reduced intracellular drug accumulation is one of the most consistently identified features of platinum-resistant cell lines, but clinical data are limited. We assessed the effects of tissue platinum concentrations on response and survival in NSCLC. PATIENTS AND METHODS We measured total platinum concentrations by flameless atomic absorption spectrophotometry in 44 archived fresh-frozen NSCLC specimens from patients who underwent surgical resection after neoadjuvant platinum-based chemotherapy. Tissue platinum concentration was correlated with percent reduction in tumor size on post- versus prechemotherapy computed tomography scans. The relationship between tissue platinum concentration and survival was assessed by univariate and multicovariate Cox proportional hazards regression model analysis and Kaplan-Meier analysis. RESULTS Tissue platinum concentration correlated significantly with percent reduction in tumor size (P < .001). The same correlations were seen with cisplatin, carboplatin, and all histology subgroups. Furthermore, there was no significant impact of potential variables such as number of cycles and time lapse from last chemotherapy on platinum concentration. Patients with higher platinum concentration had longer time to recurrence (P = .034), progression-free survival (P = .018), and overall survival (P = .005) in the multicovariate Cox model analysis after adjusting for number of cycles. CONCLUSION This clinical study established a relationship between tissue platinum concentration and response in NSCLC. It suggests that reduced platinum accumulation might be an important mechanism of platinum resistance in the clinical setting. Further studies investigating factors that modulate intracellular platinum concentration are warranted.

Synthesis and antitumor activity of ammine/amine platinum(II) and (IV) complexes

Dimeric platinum complexes, [Pt(RNH2)I2]2 (where R = H, methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl), have been synthesized by reactions of diiodoplatinum compounds with perchloric acid in water/ethanol solutions. The dimerization varies from several hours to a few days depending upon the length of the carbon chain in the alkylamines and the process can be conveniently monitored by 195Pt NMR spectroscopy. All these dimers exhibit two closely separated resonances around -4000 ppm (vs K2PtCl4 at -1620 ppm) in dimethylformamide. Reactions of [Pt(NH3)I2]2 with alkylamines do not yield the desired mixed ammine/amine complexes, which are obtained subsequently by treatment of the alkylamine dimer [Pt(RNH2)I2]2 with ammonium hydroxide in water. By using this latter procedure, a novel class of ammine/amine platinum complexes of the type PtII(NH3)(RNH2)Cl2, PtIV(NH3)(RNH2)X2A2, and PtIV(NH3)(RNH2)(CBDCA)A2.H2O, where X2 = chloro or 1,1-cyclobutanedicarboxylato (CBDCA), A = OH, Cl, or OCOCH3, have been synthesized and characterized by elemental analysis, infrared, and 195Pt NMR spectroscopic techniques. The alicyclic ammine/amine Pt(II) complexes, where R is C3-C6 were selected as representative of the class to undergo antitumor evaluations. The compounds had excellent activity against murine leukemic L1210/0 cells with cyclobutylamine-, cyclopentylamine- and cyclohexylamine-containing complexes demonstrating cytotoxicity superior to that of the clinically established cisplatin.

Synthesis, characterization and cytotoxicity of new platinum(IV) axial carboxylate complexes: crystal structure of potential antitumor agent [PtIV(trans-1R,2R-diaminocyclohexane)trans(acetate)2Cl2]

A series of new platinum(IV) complexes of the type [PtIV(DACH)trans(L)2Cl2] (where DACH = trans-1R,2R-diaminocyclohexane, and L = acetate, propionate, butyrate, valerate, hexanoate, or heptanoate) bearing the carboxylate groups in the axial positions have been synthesized and characterized by elemental analysis, IR, and 195Pt NMR spectroscopy. The crystal structure of the analogue [PtIV(DACH)trans(acetate)2Cl2] was determined by single crystal X-ray diffraction method. There were two crystallographically independent molecules, both of which lie on crystallographic two-fold axes. The bond lengths and bond angles of both the molecules were the same within the experimental error. The compound crystallizes in the monoclinic space group C2, with a = 11.180(2) A, b = 14.736(3) A, c = 10.644(2) A, beta = 112.38(3) degrees, Z = 4 and R = 0.0336, based upon a total of 1648 collected reflections. In this complex, the platinum had a slightly distorted octahedron geometry owing to the presence of a geometrically strained five-member ring. The two adjacent corners of the platinum plane were occupied by the two amino nitrogens of DACH, whereas the other two equatorial positions were occupied by two chloride ions. The remaining two axial positions were occupied by the oxygens of acetate ligands. The DACH ring was in a chair configuration. An intricate network of intermolecular hydrogen bonds held the crystal lattice together. These analogues were evaluated in vitro and demonstrated cytotoxic activity against the human ovarian 2008 tumor cell line (IC50 = 0.001-0.06 microM). Structure-activity study revealed that activity was highest for the analogue where L = butyrate.