Effects Of DTIC Research Articles

Combined Therapy with Dacarbazine and Hyperthermia Induces Cytotoxicity in A375 and MNT-1 Melanoma Cells.

Melanoma is a drug-resistant cancer, representing a serious challenge in cancer treatment. Dacarbazine (DTIC) is the standard drug in metastatic melanoma treatment, despite the poor results. Hyperthermia has been proven to potentiate chemotherapy. Hence, this work analyzed the combined action of hyperthermia and DTIC on A375 and MNT-1 cell lines. First, temperatures between 40 °C and 45 °C were tested. The effect of DTIC on cell viability was also investigated after exposures of 24, 48, and 72 h. Then, cells were exposed to 43 °C and to the respective DTIC IC10 or IC20 of each time exposure. Overall, hyperthermia reduced cell viability, however, 45 °C caused an excessive cell death (>90%). Combinational treatment revealed that hyperthermia potentiates DTIC’s effect, but it is dependent on the concentration and temperature used. Also, it has different mechanisms from the treatments alone, delaying A375 cells at the G2/M phase and MNT-1 cells at the S and G2/M phases. Intracellular reactive oxygen species (ROS) levels increased after treatment with hyperthermia, but the combined treatment showed no additional differences. Also, hyperthermia highly increased the number of A375 early apoptotic cells. These results suggest that combining hyperthermia and DTIC should be more explored to improve melanoma treatment.

Effect of dacarbazine on CD44 in live melanoma cells as measured by atomic force microscopy-based nanoscopy.

CD44 ligand–receptor interactions are known to be involved in regulating cell migration and tumor cell metastasis. High expression levels of CD44 correlate with a poor prognosis of melanoma patients. In order to understand not only the mechanistic basis for dacarbazine (DTIC)-based melanoma treatment but also the reason for the poor prognosis of melanoma patients treated with DTIC, dynamic force spectroscopy was used to structurally map single native CD44-coupled receptors on the surface of melanoma cells. The effect of DTIC treatment was quantified by the dynamic binding strength and the ligand-binding free-energy landscape. The results demonstrated no obvious effect of DTIC on the unbinding force between CD44 ligand and its receptor, even when the CD44 nanodomains were reduced significantly. However, DTIC did perturb the kinetic and thermodynamic interactions of the CD44 ligand–receptor, with a resultant greater dissociation rate, lower affinity, lower binding free energy, and a narrower energy valley for the free-energy landscape. For cells treated with 25 and 75 μg/mL DTIC for 24 hours, the dissociation constant for CD44 increased 9- and 70-fold, respectively. The CD44 ligand binding free energy decreased from 9.94 for untreated cells to 8.65 and 7.39 kcal/mol for DTIC-treated cells, which indicated that the CD44 ligand–receptor complexes on DTIC-treated melanoma cells were less stable than on untreated cells. However, affinity remained in the micromolar range, rather than the millimolar range associated with nonaffinity ligands. Hence, the CD44 receptor could still be activated, resulting in intracellular signaling that could trigger a cellular response. These results demonstrate DTIC perturbs, but not completely inhibits, the binding of CD44 ligand to membrane receptors, suggesting a basis for the poor prognosis associated with DTIC treatment of melanoma. Overall, atomic force microscopy-based nanoscopic methods offer thermodynamic and kinetic insight into the effect of DTIC on the CD44 ligand-binding process.

Inhibitory effect of CDK2 gene silencing combined with dacarbazine on the growth of B16-F1 melanoma

Objective To evaluate the antitumor effect of dacarbazine (DTIC) on B16-F1 melanoma after CDK2 gene silencing. Methods Cultured B16-F1 melanoma cells were divided into 4 groups: control group receiving no treatment, CDK2-shRNA group infected with a recombinant lentivirus pUL-CDK2-shRNA, DTIC group cultured in 96-well plates followed 12 hours later by the treatment with 250 μmol/L DTIC, CDK2-shRNA+ DTIC group infected with pUL-CDK2-shRNA followed 12 hours later by the treatment with 250 μmol/L DTIC. MTT assay was performed to evaluate the growth inhibition of B16-F1 melanoma cells, and coefficient of drug interaction (CDI) was calculated. AnnexinV-FITC/PI double staining was conducted to detect cell apoptosis. C57BL/6 mice were subcutaneously injected with B16-F1 cells at exponential growth phase into the right groin to establish melanoma-bearing mouse models.Twenty mouse models were randomly and equally divided into 4 groups: control mouse group injected with phosphate-buffered solution (PBS) into tumors, CDK2-shRNA mouse group injected with pUL-CDK2-shRNA into tumors, DTIC mouse group injected with DTIC into the abdominal cavity, and CDK2-shRNA+ DTIC mouse group treated with pUL-CDK2-shRNA and DTIC. The animal experiment lasted 18 days, and the tumor growth curve was drawn. After 18-day treatment, all the mice were sacrificed, and tumors were isolated and weighed. The tumor growth inhibition rate was calculated, and the tumor cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) . Results After 72-hour culture, compared with the control group, the CDK2-shRNA group, DTIC group, and CDK2-shRNA+ DTIC group showed significantly decreased relative cell survival rates (40.6% ± 2.8%, 45.2% ± 3.7%, 28.7% ± 2.1%, respectively; F= 458.04, P < 0.05) , but significantly increased cell apoptosis rates (25.1% ± 3.3%, 15.6% ± 2.2%, 45.6% ± 3.5%, respectively; F= 115.46, P < 0.05) . Additionally, CDK2-shRNA+ DTIC group showed significantly lower relative cell survival rates (P < 0.01) , but higher cell apoptosis rates (P < 0.01) compared with the DTIC group. The CDI value was less than 0.7. On the sixth day after the in vivo treatment, the tumor volumes in the control mouse group, CDK2-shRNA mouse group, DTIC mouse group, and CDK2-shRNA+ DTIC mouse group were (185.44 ± 68.97) mm3, (83.91 ± 14.33) mm3, (123.70 ± 20.85) mm3, and (34.54 ± 10.72) mm3 respectively. From then on, the CDK2-shRNA mouse group, DTIC mouse group, and CDK2-shRNA+ DTIC mouse group showed significantly decreased tumor growth rates compared with the control mouse group (F= 11.819, P < 0.05) , and the tumor growth rate was significantly lower in the CDK2-shRNA+ DTIC mouse group than in the DTIC mouse group (P= 0.04) . The calculated tumor growth inhibition rates in the CDK2-shRNA mouse group, DTIC mouse group and CDK2-shRNA+ DTIC mouse group were 52.2%, 41.2% and 86.4% respectively. Compared with the control mouse group, the CDK2-shRNA mouse group, DTIC mouse group, and CDK2-shRNA+ DTIC mouse group showed significantly increased tumor cell apoptosis indice (32.93% ± 3.72%, 21.62% ± 3.54%, 63.29% ± 4.74% respectively; F= 222.25, P < 0.05) . Moreover, the tumor cell apoptosis index was significantly higher in the CDK2-shRNA+ DTIC mouse group than in the DTIC mouse group (P < 0.01) . Conclusion CDK2 gene silencing can enhance the inhibitory effect of DTIC on the growth of melanoma, and show a synergistic effect with DTIC, likely by increasing the apoptosis of tumor cells. Key words: Melanoma; Dacarbazine; Cyclin-dependent kinase 2; Gene silencing; Apoptosis

Acute toxic effects of single dose dacarbazine: hematological and histological changes in an animal model

Treatment of advanced soft tissue sarcoma usually includes dacarbazine (DTIC), an alkylating agent that methylates DNA and is active during all phases of the cell cycle. Common side effects of DTIC include nausea, vomiting, impaired liver and kidney function, myelosuppression, and pneumonia. There are no accounts, however, of histological and hematological changes caused by DTIC. We investigated acute hematological and morphological changes in different organs and in tumors that were caused by a single dose of DTIC. Adult Syrian golden hamsters were inoculated with a suspension of tumorigenic baby hamster kidney (BHK) cells by subcutaneous injection. On day 14 after inoculation, doses of 1.4, 1.6, 1.8 or 2.0 g/m2 DTIC were injected intraperitoneally into the hamsters. Hamsters in the control group were injected with physiological saline in the same way. Seven days after drug or saline injection the animals were sacrificed and samples of blood, heart, kidney, liver, lungs, spleen, small intestine and tumor were excised, processed and analyzed. Mitoses were counted using an ocular extension with engraved frame. Anemia, thrombocytopenia and leukocytosis were found in the control group of hamsters with fibrosarcoma, whereas animals with fibrosarcoma treated with DTIC developed anemia, thrombocytopenia and leukopenia. Severe pneumonia and moderate hepatitis were detected in all DTIC treated groups. Effects of DTIC on tumor cells included rounding and enlargement of nuclei and rarefaction of chromatin. The number of mitoses was reduced with increasing doses of DTIC. Hepatitis, myelosuppression, pneumonia, and dose-related inhibition of tumor cell proliferation were observed after a single dose of DTIC.

Lenvatinib combined with dacarbazine versus dacarbazine alone as first-line treatment in patients with stage IV melanoma.

9027 Background: Lenvatinib (E7080; LEN) is an oral, receptor tyrosine kinase inhibitor targeting VEGFR1-3, FGFR1-4, RET, KIT and PDGFRβ . In a phase I study qd dosing of &gt;/= 20mg LEN demonstrated PRs and prolonged SD in patients (pts) with advanced melanoma. Dacarbazine (DTIC) upregulates the proangiogenic factor VEGF and has been shown to confer resistance in melanoma cell lines (Lev, JCO 2004; 22:2092-2100). Treatment of melanoma pts with LEN + DTIC may potentiate the therapeutic effects of DTIC. Methods: This was a phase II study in pts with metastatic melanoma randomized 1:1 to receive LEN (20 mg QD) + DTIC (1000 mg/m2 Q 21 d) or DTIC (1000 mg/m2Q 21 d). Pts were stratified by LDH level and stage IV subclass. Eligible pts were ECOG PS 0/1 and had no prior systemic therapies. BRAF status was determined from circulating tumor DNA. The primary endpoint was PFS by independent assessment. Results: In a modified ITT analysis,a total of 78 of 81 pts were evaluable for efficacy; 59% were male, 59% were Stage IV M1c, 22% had elevated LDH, 29% had prior adjuvant therapy and 49% BRAF wild-type (wt). Most common AEs in the LEN + DTIC arm were hypertension (48%), nausea (38%), constipation (33%), and diarrhea (31%). Most common Grade 3/4 AEs in LEN + DTIC were hypertension (26%) and neutropenia (10%). There were no deaths due to an AE. Median PFS increased in LEN + DTIC compared to DTIC alone (see Table). An improvement in median PFS was observed in BRAFwt pts on the combination. Conclusions: A 2.7-fold increase in the median PFS was observed in pts administered LEN + DTIC compared to single agent DTIC. A 2.5 fold increase in the median PFS was observed in the combination arm in pts with BRAFwt melanoma. The AE profile observed with the LEN + DTIC was consistent with observed LEN monotherapy studies. These data suggest further evaluation of LEN + DTIC in BRAFwt melanoma is warranted. Clinical trial information: NCT01133977. [Table: see text]

Synthesis and theoretical conformational studies of gonadotropin-releasing hormone analogs containing tetrahydroisoquinoline carboxylic acid

A series of analogs of gonadotropin-releasing hormone (GnRH) containing the conformationally restrictive residue tetrahydroisoquinoline carboxylic acid (Tic) or its nonrestricted parent phenylalanine were synthesized and evaluated for anti-ovulatory activity in the rat. The series, based on the potent linear parent compound Ac-DNal 1-DCpa 2-DPal 3-Ser 4-Tyr 5-DPal 6-Leu 7-Arg 8-Pro 9- DAla 10-NH 2, included L-Tic in positions 1, 2, 3, 7 and 9, D-Tic in positions 1, 2, 3, 6 and 9, or D-Phe in positions 2 and 3 for comparison. The most potent analog in this series, with D-Tic in position 6, fully inhibited ovulation at 2.5 μg compared to near complete inhibition at 0.5 μg for the parent compound. A theoretical analysis of the conformational restrictions imposed on mainchain and sidechain torsional angles by the incorporation of Tic was conducted in vacuo using molecular mechanics techniques. Using cyclo(4–10)-[Ac-Δ 3Pro 1,DCpa 2,DTrp 3,Asp 4,DNal 6,Dpr 10]-GnRH as a template conformer for which NMR conformational data is available, it was found that the potency of the different analogs correlated with the strain energy required to deform the mainchain and backbone angles of residues to values which would be expected if Tic were present and if the analog assumed the same solution structure. In particular, the effect of DTic at position 6 is to maintain the type II′ beta-turn involving residues 5–8 found in active GnRH analogs.

Effects of DTIC, DM-COOK and ICRF-159 on the number of circulating Lewis lung carcinoma cells detected by flow cytometry.

Circulating tumor cells can be detected by means of flow cytometry in the blood of mice bearing i.m. Lewis lung carcinoma. This technique can be applied in the case of aneuploid tumors and does not require either concentration of nucleated cells or other processing of the blood samples. It offers the advantages of simplicity and speed and allows quantitative measurement of the number of circulating tumor cells. It can be applied to the study of the effects of drug treatment on the number of circulating tumor cells, for those drugs which do not cause alterations in the nuclear DNA content of normal diploid blood cells. The number of circulating tumor cells determined by flow cytometry is markedly reduced by treatment with ICRF-159, by dimethyltriazene DM-COOK, and also by its clinically used analog, DTIC.

The antitumour agent 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC) inhibits rat liver cAMP phosphodiesterase and amplifies hormone effects in hepatocytes and hepatoma cells

The antitumour agent 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) was found to inhibit competitively the low-Km cyclic AMP phosphodiesterase activity in an ammonium-sulphate-precipitable fraction of the 2,000g supernatant of rat liver. With substrate concentration at 0.25 microM, I50 was 790 microM for DTIC and 350 microM for theophylline. DTIC at 2 mM more than doubled the cAMP response to glucagon in hepatocytes and to adrenaline in MH1C1 hepatoma cells, indicating that it also exerts its inhibitory effect on the phosphodiesterase in intact cells. The possible contribution of the phosphodiesterase inhibition to the growth-inhibitory and cytotoxic effects of DTIC is discussed.