Targeted therapy against Bcl-2-related proteins in breast cancer cells

INTRODUCTION: Bcl-2 and Bcl-xL confer resistance to apoptosis, thereby reducing the effectiveness of chemotherapy. We examined the relationship between the expression of Bcl-2 and Bcl-xL and chemosensitivity of breast cancer cells, with the aim of developing specific targeted therapy. METHODS: Four human breast cancer cell lines were examined, and the effects of antisense (AS) Bcl-2 and AS Bcl-xL phosphorothioate oligodeoxynucleotides (ODNs) on chemosensitivity were tested in vitro and in vivo. Chemosensitivity was evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, and the antitumor effect was assessed in vivo by the success of xenograft transplantation into athymic mice. RESULTS: Treatment with AS Bcl-2 and Bcl-xL ODNs resulted in a sequence-specific decrease in protein expression, compared with controls. Treatment of BT-474, ZR-75-1, and MDA-MB-231 cells with AS Bcl-2 increased chemosensitivity to doxorubicin (DOX), mitomycin C (MMC), paclitaxel (TXL), and docetaxel (TXT). Transfection of the Bcl-2 gene into MDA-MB-453 cells decreased sensitivity to DOX and MMC. Treatment of MDA-MB-231, BT-474, and ZR-75-1 cells with AS Bcl-xL increased chemosensitivity to DOX, MMC and taxanes to a smaller extent than AS Bcl-2. This occurred in the setting of increased Bax and cleaved poly(ADP-ribose) polymerase, as well as decreased Bcl-2 and pAkt. AS Bcl-2 ODNs induced splenomegaly in association with increased serum IL-12, which was attenuated by methylation of the CpG motifs of AS Bcl-2; however, methylated CpG failed to negate the increased antitumor effect of AS Bcl-2. Bcl-2 and Bcl-xL, to a smaller extent, are major determinants of chemosensitivity in breast cancer cells. CONCLUSION: Targeted therapy against Bcl-2 protein with the use of AS ODNs might enhance the effects of chemotherapy in patients with breast cancer

Tumor-Specific Synergistic Therapy of Mitomycin C : Modulation of Bioreductive Activation

The bioreductive activation of mitomycin C (MMC) has been investigated using 10 human cancer cell lines. Except for 2 lines (COLO201 and COLO320DM), the cellular NAD(P)H: quinone oxidoreductase (DTD) activities correlated well with MMC-induced DNA damage and cytotoxicity. In addition, when the DTD activity was inhibited with 50 mM dicoumarol, the MMC activity decreased significantly. On the other hand, no correlation between the NADPH cytochrome P450 reductase (P450) activity and MMC efficacy was observed. We postulate that two electron reduction by DTD may be more important in MMC activation than one electron reduction by P450. The DTD-mediated metabolism was pH-dependent. In a nude mouse experimental model, the pH in the tumor decreased under hyperglycemic conditions due to unique glycolysis. The administration of m-iodobenzyl-guanidine (MIBG) enhanced the decrease in the pH of the tumor without affecting the pH of normal tissue (liver). It also significantly increased the antitumor activity of MMC. However, this biochemical modulation had no effect in the COLO201 and COLO320DM cells. Other mechanisms may be involved in the regulation of MMC activity in these cells. In conclusion, DTD may be an important target of MMC. Biochemical modulation using MIBG and glucose may selectively enhance the activity of MMC within cancer cells.This work was presented in part at the 95th and 96th Annual Meetings of the Japan Society for Surgery, Nagoya, 1994, and Makuhari, 1996

DT-diaphorase as a Target Enzyme for Biochemical Modulation of Mitomycin C

We studied a selective enhancement of the mitomycin C (MMC)-induced antitumor effect focusing on the intracellular metabolism by NAD(P)H:quinone oxidoreductase (DT-diaphorase, DTD). The level of cellular DTD activity related well to the degree of MMC-induced DNA total cross links and cell growth inhibition in human cancer cell lines, KB, PH101, SH101 and K562. A DTD inhibitor, dicoumarol (DIC) or flavin adenine dinucleotide (FAD), inhibited the MMC-induced DNA damage and cytotoxicity at a non-toxic concentration. The DTD-mediated MMC activation was pH-dependent, and highest at pH 6 and lowest at pH 8. Although an inverse relationship appeared to exist between DTD activity and MMC efficacy in human xenografts implanted into nude mice and 9 fresh human tumor specimens, the investigation in 3 culture cells, HEC-46, HCC-48 and HCC-50, established from those xenografts, showed that DTD activated MMC in a pH-dependent manner as well as the other cell lines. Significant tumor pH reduction from 7.1 to 6.7 by continuous glucose infusion also increased the MMC-induced tumor growth inhibition in the human tumor xenografts. Thus, we conclude that bioreductive activation by DTD in a pH-dependent manner may be of key importance in the MMC-induced antitumor effect and that an increased MMC efficacy at a reduced pH caused by hyperglycemia may be applied to clinical use as a new manipulation for a biochemical modulation of MMC.This work was presented in part at the 40th Annual Meeting of Japan Soc. for Chemotherapy, Nagoya, 1992, the 18th International Congress of Chemotherapy, Stockholm, 1993 and the 52nd Annual Meeting of Japan Cancer Assoc., Sendai, 1993

Circadian Variation of 5-Fluorouracil and Cis-Platinum Toxicity in Mice

Circadian variations of organ specific and lethal toxicities were investigated in fem ale C3H mice following intraperitoneal injection(s) of 5-fluorouracil (5-FU) and/or cis-platinum (CDDP) with different doses at one of the four equidistant time points, viz., 3, 9, 15 and 21 HALO (Hours After Light On). Lethality, peripheral WBC count, spleen size, femoral bone marrow cell population and changes in body weight were analyzed. A single injection of 5-FU (300 mg/kg) showed 45% mortality with 3 HALO and 15% with 9 HALO treatment (p<0.05); that with CDDP (16 mg/kg) was 80% in 3 HALO and 10% in 9 HALO group (p<0.01). Peripheral WBC count on the 4th post-treatment day showed that drug induced leukopenia was less severe with 15 HALO dosing compared to the other HALO points (p<0.01). The reduction in spleen size following treatment was maximum in 3 HALO treated group with both drugs (p<0.01). The combination therapy of these two drugs also showed circadian variation of toxicity. The results suggest that the toxic effects of chemotherapy are dosing time dependent

Clinical Evaluation of Low-dose Cisplatin and 5-Fluorouracil as Adjuvant Chemoradiotherapy for Advanced Squamous Cell Carcinoma of the Esophagus

The aim of this retrospective study was to evaluate the effectiveness of low-dose cisplatin and 5-fluorouracil (low-dose FP) as an adjuvant chemoradiotherapy for resected advanced squamous cell carcinoma of the esophagus. From 1994 to 1999, 57 patients who showed an invasion of the tumor over the muscularis propria (T2-T4), regional lymph node metastasis (Nl), and no distant metastasis (MO) were enrolled in this analysis. Postoperative chemoradiotherapy (CRT group) was performed on 14 of the patients, and they were compared to the patients who underwent surgery alone (S group) using the matched pair algorithm. In the CRT group, chemotherapy of low-dose FP was combined with concurrent radiotherapy after the esophagectomy. A side-effect of severe dysphagia (NCI-CTC Grade 3) was observed in 4 patients (28.6%) and leukocytopenia in 1 patient (7.1%) among the CRT group. The overall survival rate of the CRT group and matched S group were 35. 7% and 28.5% at 5 years, respectively, with no significant difference. In the CRT group, 7 of 14 patients (50%) had a recurrence. The recurrence rate was slightly lower than in the S group (57%), with no significant difference. This combined chemoradiotherapy using low-dose FP did not improve the prognosis of patients with resected advanced esophageal carcinoma