Bleomycin induced changes in growth and cell kinetics of a heterotransplanted squamous cell carcinoma of the head and neck

Bleomycin (BLM) is often used against squamous cell carcinomas in combination with other drugs in phase-specific schedules since it has been considered a synchronizing agent. However, the reported effects on the cell cycle are contradictory. In the present study a poorly differentiated squamous cell carcinoma of the head and neck, heterotransplanted to nude mice was used for evaluation of BLM induced suppression of growth, and, measured with flow cytometric technique, cell cycle traverse and cell cycle phase distribution. BLM was administered once daily for four days. The dose-response curve exhibited a plateau-phase beginning at a total dose of 200 mg BLM/kg. A single dose of 75 mg BLM/kg caused a transient accumulation of cells in G0 + G1 phase and was followed by a synchronized passage of cells through the cell cycle. However, the induced perturbations were small. Cell kinetic analysis of tumours treated with repeated doses of BLM did not result in increased cell cycle perturbations. Thus, the present study could not confirm any major cell synchronizing effect of BLM on poorly differentiated heterotransplanted human squamous cell carcinoma.     

Changes in growth pattern of human squamous-cell carcinomas of the head and neck during serial passages in nude mice

Six human squamous-cell carcinomas of the head and neck heterotransplanted to nude mice were studied with respect to changes in tumour volume doubling time (DT), cell cycle phase distribution and clonal composition during serial passages. The tumours exhibited a statistically significant decrease in DT between the first and third or fourth passages towards a constant value. The changes in DT are likely to depend on a reduction of cell loss factor. Five of the original tumours and serially passed heterotransplants were analyzed with flow cytometry. Two tumours were diploid and remained so after heterotransplantation. Three tumours had one diploid and one aneuploid cell population. At heterotransplantation there was a selection of clones and only the aneuploid cell populations could be recovered from the heterotransplants in the first as well as in later passages.     

Antitumor activity of cis-dichlorodiammineplatinum(II) and its effect on cell cycle progression

Antitumor activity of cis-dichlorodiammineplatinum(II) (cisplatin) on various mouse transplantable tumors was investigated. Cisplatin was active against a wide variety of the following tumor systems: L1210 leukemia, P388 leukemia, B16 melanoma, colon tumor 38, Ehrlich ascites and solid carcinoma, WHT squamous cell carcinoma, and human stomach cancer G/S heterotransplanted in nude mice. From the comparison of growth inhibitory effect by cisplatin with various other antitumor agents in cultured Ehrlich ascites carcinoma cells, cisplatin was found to be mainly a concentration depending drug, but also time depending, so that it was identified as a type Ib class drug proposed by Shimoyama. Effect of cisplatin on the cell cycle progression of Ehrlich ascites carcinoma cells in mice was studied by flow cytometry of DNA. At an early stage after administration of cisplatin, cell cycle progression was delayed in S phase and blocked in G2 phase. With the elapse of time block in G1 phase or G1-S boundary was observed and the cell population, partially synchronized in G1 phase or G1-S boundary, progressed slowly through S phase to be blocked in G2 phase finally.     

Histopathological characteristics predictive for growth of squamous cell carcinomas of the head and neck heterotransplanted to nude mice

Human tumours heterotransplanted to nude mice vary both with respect to take rate and the rate of tumour line establishment, according to their histological type and between primary and recurrent tumours. The acceptance rate of squamous cell carcinomas of the head and neck is also thought to vary, according to their degree of differentiation and their site of origin. This provided the basis of the present analysis of different tumour characteristics predicting growth of squamous cell carcinomas of the head and neck. Eighty-five attempted heterotransplantations were analysed with respect to T-stage, site of origin, degree of differentiation, and a histopathological malignancy grading score based upon four tumour variables and four tumour-host variables. Of the 85 attempted transplantations, first passage was successful in 24 cases (28%), of which sixteen (19% of 85) resulted in established tumour lines. The frequency of established tumour lines was higher among T4 and poorly differentiated tumours (33% and 22%, respectively), than among less advanced or more differentiated tumours. Tumour size was a more crucial factor than degree of differentiation, and tumours of the oral cavity grew less readily than those from other sites. The take rate was strongly correlated to malignancy grading scores, increasing as they increased. The most predictive information was provided by tumour-host variables, of which vascular invasion and lack of lymphocytic response were the most important in this respect.     

Cell cycle effects of gemcitabine.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, or dFdC) is a promising anticancer agent with demonstrated clinical activity in solid tumours currently undergoing clinical trials. Despite extensive studies on the biochemical mechanism of action, cell cycle perturbations induced by dFdC have not yet been thoroughly investigated, apart from the expected inhibition of DNA synthesis. The aim of our study was to clarify whether cell population kinetics is a vital factor in the cytotoxicity of dFdC in single or repeated treatments and in the dFdC-cisplatin combination. Ovarian cancer cells growing in vitro were treated with dFdC for 1 hr in a range of concentrations from 10 nM to 10 microM. Cell kinetics was investigated by DNA-bromodeoxyuridine flow cytometry, using different experimental protocols to measure either the time course of DNA-synthesis inhibition or the fate of cells in G(1), S or G(2)M at the time of dFdC treatment or 24 hr later. A modified sulforhodamine B test was used to assess the growth inhibition caused by dFdC given alone or with cisplatin. Although dFdC promptly inhibited DNA synthesis, cytotoxicity on proliferating cells was not specific for cells initially in the S phase. DNA synthesis was restored after a G(1) block of variable, dose-dependent length, but recycling cells were intercepted at the subsequent checkpoints, resulting in delays in the G(2)M and G(1) phases. The activity of repeated treatment with dFdC + dFdC or dFdC + cisplatin was highly dependent on the interval length between them. These results suggest that the kinetics of cell recycling from a first dFdC treatment strongly affects the outcome of a second treatment with either dFdC itself or cisplatin.     

Propagation of a poorly differentiated human pulmonary adenocarcinoma in nude athymic rats

Nude athymic rats of Rowett genetic background were injected subcutaneously with cells from a poorly differentiated human pulmonary adenocarcinoma, following propagation in nude mice. The tumour cells were obtained by thoracentesis and by pleural biopsy during thoracoscopy. So far, we have heterotransplanted 13 different malignant human tumours including malignant mesothelioma, pulmonary adenocarcinoma, malignant melanoma and malignant lymphoma. Subcutaneous tumour growth was seen in all inoculated animals, with an exponential tumour growth pattern and with tumour volume doubling times of approximately 8 days. After two initial passages in nude athymic mice of BALB/c genetic background, the human tumour so far has undergone 9 passages in athymic rats, i.e. collection of tumour material by biopsy from the preceding tumour-bearing rat generation, processing and inoculation of tumour brei with subsequent tumour growth have been repeated 8 times. Four-to-8-week-old rats of both sexes were used throughout and tumour growth was controlled by palpation twice a week. Routine histopathological sections were prepared from the tumour at various passages to assess similarity to the original tumour in the patient. The growth pattern of the xenografts remained similar at all passages just as the remarkable similarity of the heterotransplanted tumours to the original adenocarcinoma was retained at all passages. No spontaneous regressions of heterotransplanted tumours could be demonstrated. Electron microscopical analysis revealed numerous blunt microvilli and lumina partly filled with conglomerates of mucigen granules and small glycocalyceal bodies associated with external superficial microvilli. Scantiness of dark secretory granules together with free and membrane-bound polyribosomes were seen in the cytoplasm. We believe that the nude athymic rat is a valuable research tool and that the permanently transplantable human tumour reported here could be of value in delineating further the mechanisms for tumour take, growth and control.     

Changes of proliferation kinetics after X-irradiation of a human malignant melanoma grown in nude mice

A human malignant melanoma grown in nude mice was exposed to single-dose X-irradiation and the effect on the proliferation kinetics was investigated by two methods. Flow cytometric DNA analysis was performed on tumour tissue obtained by sequential fine-needle aspirations after the treatment to monitor the initial cell cycle distribution changes. The technique of labelled mitoses was used to examine the kinetics of the tumours during regrowth. The results showed that the treatment initially induced a partial synchronization of small fractions of cells accumulated in the G₂ phase of the cell cycle and a dose-dependent decrease of the cell generation time due to a shortening of the G₂ duration time during regrowth of the tumours. Furthermore, it was shown that the calculated values of growth fraction and cell loss factor became unreliable because the tumours contained a dose-related increasing proportion of radiation-inactivated tumour cells.     

NK105, a paclitaxel-incorporating micellar nanoparticle, is a more potent radiosensitising agent compared to free paclitaxel

NK105 is a micellar nanoparticle formulation designed to enhance the delivery of paclitaxel (PTX) to solid tumours. It has been reported to exert antitumour activity in vivo and to have reduced neurotoxicity as compared to that of free PTX. The purpose of this study was to investigate the radiosensitising effect of NK105 in comparison with that of PTX. Lewis lung carcinoma (LLC)-bearing mice were administered a single intravenous (i.v.) injection of PTX or NK105; 24 h after the drug administration, a proportion of the mice received radiation to the tumour site or lung fields. Then, the antitumour activity and lung toxicity were evaluated. In one subset of mice, the tumours were excised and specimens were prepared for analysis of the cell cycle distribution by flow cytometry. Combined NK105 treatment with radiation yielded significant superior antitumour activity as compared to combined PTX treatment with radiation (P=0.0277). On the other hand, a histopathological study of lung sections revealed no significant difference in histopathological changes between mice treated with PTX and radiation and those treated with NK105 and radiation. Flow-cytometric analysis showed that NK105-treated LLC tumour cells showed more severe arrest at the G2/M phase as compared to PTX-treated tumour cells. The superior radiosensitising activity of NK105 was thus considered to be attributable to the more severe cell cycle arrest at the G2/M phase induced by NK105 as compared to that induced by free PTX. The present study results suggest that further clinical trials are warranted to determine the efficacy and feasibility of combined NK105 therapy with radiation.     

Radioimmunotherapy of human head and neck squamous cell carcinoma xenografts with 131I-labelled monoclonal antibody E48 IgG.

Monoclonal antibody (MAb) E48 reacts with a 22 kD antigen exclusively expressed in squamous and transitional epithelia and their neoplastic counterparts. Radiolabelled with 99mTc, MAb E48 is capable of targeting metastatic and recurrent disease in patients with head and neck cancer. In this study, the capacity of 131I-labelled MAb E48 to eradicate xenografts of human squamous cell carcinoma of the head and neck (HNSCC) in nude mice was examined. Experimental groups received a single i.v. bolus injection of 400 microCi MAb E48 IgG (number of mice (n = 6, number of tumours (t) = 9) or 800 microCi MAb E48 IgG (n) = 5,t = 7), whereas control groups received either diluent (n = 3,t = 5), unlabelled MAb E48 IgG (n = 4,t = 5) or 800 microCi 131I-labelled isotype-matched control MAb (n = 6,t = 9). A 4.1-fold increase in the median tumour volume doubling time and regression of two out of ten tumours (20%) was observed in mice treated with 400 microCi. In mice treated with 800 microCi. In mice treated with 800 microCi, two out of seven tumours (29%) showed complete remission without regrowth during follow-up (greater than 3 months). Median tumour volume doubling time in the remaining five tumours was increased 7.8-fold. No antitumour effects were observed in mice injected with diluent, unlabelled MAb E48 or 131I-labelled control MAb. In the same xenograft model, chemotherapy with doxorubicin, 5-fluorouracil, cisplatin, bleomycin, methotrexate or 2',2'-difluorodeoxycytidine yielded a less profound effect on tumour volume doubling time. Increases in tumour volume doubling time with these chemotherapeutic agents were 4, 2.2, 2.1, 1.7, 0, and 2.6 respectively. Moreover, no cures were observed with any of these chemotherapeutic agents. From the tissue distribution of 800 microCi MAb E48, the absorbed cumulative radiation doses of tumour and various organs were calculated using the trapezoid integration method for the area under the curve. To tumour xenografts, 12,170 cGy was delivered, blood received 2,984 cGy, whereas in every other tissue the accumulated dose was less than 6% of the dose delivered to tumour. These data, describing the first radiolabelled MAb with therapeutic efficacy against HNSCC, suggest radioimmunotherapy with MAb E48 to be a potential therapeutic modality for the treatment of head and neck cancer.     

Endocrine sensitivity of the receptor-positive T61 human breast carcinoma serially grown in nude mice

A study was made on the effect of ovariectomy, 17 beta-oestradiol, and tamoxifen on the oestrogen and progesterone receptor-positive T61 human breast carcinoma grown in nude mice. The effect of the treatment was evaluated by the specific growth delay calculated on the basis of Gompertz growth curves, and by the changes in the cell cycle distribution monitored by flow cytometric DNA analysis. The results demonstrated that both oestradiol and tamoxifen induced a temporary growth delay, whereas ovariectomy of the host had no effect on the growth of the tumour. The oestradiol-induced tumour growth delay was accompanied by a decrease in the G1 fraction, an accumulation of cells in the S-phase, and polyploidy, whereas neither treatment with tamoxifen nor ovariectomy influenced cell cycle distribution. The results indicate that oestradiol and tamoxifen have different mechanisms of action. In addition, they were interpreted as indicating different mechanisms regulating ovarian-dependent tumour growth, on the one hand, and oestrogen and tamoxifen-induced tumour growth inhibition, on the other. The results support the view that the presence of receptors may be of importance but is not a sufficiently clear marker to allow prediction of the endocrine sensitivity of individual breast tumours.     

A novel immunohistochemical method for estimating cell cycle phase distribution in ovarian serous neoplasms: implications for the histopathological assessment of paraffin-embedded specimens

We have investigated whether immunohistochemical markers can identify differences in cell cycle phase distribution in ovarian serous neoplasms, including borderline tumours of different grades. Sections of normal ovary (n=18), serous cystadenoma (n=21), borderline serous tumours (n=21) and serous cystadenocarcinoma (n=15) were analysed by immunohistochemistry using markers of cell cycle entry (Mcm-2) and cell cycle phase, including cyclin D1 (mid-to-late G1), cyclin A (S phase), cyclin B1 (G2 phase) and phosphohistone H3 (mitosis). Double-labelling confocal microscopy confirmed marker phase specificity and phase estimations were corroborated by flow cytometry. On progression from normal ovary through serous cystadenoma and borderline tumours to cystadenocarcinomas, expression of Mcm-2 (P<0.0001), cyclin D1 (P=0.002), cyclin A (P<0.0001), cyclin B1 (P<0.0001) and phosphohistone H3 (P<0.0001) increased, paralleled by an increase in the S-phase fraction (cyclin A : Mcm-2 ratio; P=0.002). Borderline tumours of increasing grade also showed increased Mcm-2 and cyclin A expression, together with an increase in the S-phase fraction. Immunohistochemistry can be used to estimate cell cycle phase distribution in ovarian serous neoplasms, giving results similar to flow cytometric analysis and enabling direct assessment of tumour heterogeneity. Immunohistochemical estimates of the S-phase fraction may identify serous borderline tumours likely to exhibit malignant progression and/or select serous cystadenocarcinomas likely to respond to adjuvant therapy.     

Radiation Effects on S-Phase Duration, Labelling Index, Potential Doubling Time and DNA Distribution in Head and Neck Cancer Xenografts

The effect of irradiation on S-phase duration (Ts), labelling index (LI), potential doubling time (Tpot), and cell cycle phase distributions was determined by DNA flow cytometry in xenografted human squamous cell carcinoma of the head and neck (SCCHN). Tumours were treated with a single dose of 3 Gy, and excised at intervals over a 90-h period. Six hours before each excision the tumours were labelled in vivo with bromodeoxyuridine (BrdUrd). Although the growth rate of irradiated tumours was comparable with that of untreated controls, analysis of BrdUrd uptake revealed a transient reduction of LI and a prolongation of Ts in irradiated tumours. Maximum mean Tpot was 931 days in irradiated tumours as compared to 13 days in untreated controls. The variations in Ts, LI and Tpot all occurred within the first hours after irradiation; during the remainder of the observation time, the values of the variables did not differ from those of untreated controls. In irradiated tumours the distribution of cells according to DNA content changed significantly on three occasions during the observation period: 1) Parallel to the initial lowering of LI and prolongation of Ts there was a transient increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S and G2; 2) At 18 h, the most pronounced cell cycle phase redistribution occurred when the G0/G1 fraction decreased and the S and G2 phase fractions increased; 3) At 66 h (i.e., approximately one cell cycle later), the pattern was the same as that after 18 h. The findings suggest that the transient prolongation of DNA replication seen in SCCHN cells immediately after a single radiation dose is a symptom of DNA damage inflicted during late G1 or early S-phase, and that this disturbance in DNA synthesis is associated with the subsequent accumulation of cells in G2 phase.     

Effect of cisplatin and c-myb antisense phosphorothioate oligodeoxynucleotides combination on a human colon carcinoma cell line in vitro and in vivo.

We investigated the effect of c-myb antisense phosphorothioate oligodeoxynucleotides [(S)ODNs] and cisplatin (CDDP) combination on the human colon carcinoma cell line LoVo Dx both in vitro and in nude mice bearing LoVo Dx solid tumour. We show that antisense (S)ODN treatment decreases c-myb mRNA and protein expression, induces growth arrest in the G1 phase of the cell cycle, and inhibits cell proliferation. In vivo treatment with c-myb antisense (S)ODNs results in a reduction in tumour growth. A greater inhibition of cell proliferation in vitro and a higher increase of tumour growth inhibition and growth delay in vivo were obtained with the combination of (S)ODNs and CDDP than when the two agents were administered separately. This comparative study, using the same tumour cell line in vitro and in vivo, suggests that c-myb antisense (S)ODNs might be useful in the therapy of colon cancer in combination with antineoplastic drugs.     

C-type virus particles in human urogenital tumours after heterotransplantation into nude mice.

C-type viruses were formed in heterotransplants of 5/14 human urogenital tumours which had been serially transferred in nude mice of NIH(S) background. Except for one case in which C-type particles were present in the epithelial cells as well as the connective tissue, the viruses were only found within the stroma of the heterotransplanted tumours. Peroxidase labelling with anti-mouse serum demonstrated that the connective tissue supporting the transplanted human tumours was of mouse origin. Competition radioimmunoassays demonstrated that MuLV interspecies viral protein was present in high titre in the transplanted tumour extracts and also in extracts of 2 spontaneous mouse-tumour extracts. These data suggest that endogenous viruses of the nude mice are activated by the graft, and only subsequently infect the human tumour cells and form particles.     

The labelling index: a prognostic factor in head and neck carcinoma

The thymidine labelling index (LI), representing the percentage of cells in the DNA-synthesis phase, was measured in vitro prior to therapy in 87 patients with squamous cell carcinoma of the head and neck, who were treated between 1977 and 1982. The LI was not related to patient age, site of the tumour, clinical stage or histological grade. Overall survival was 44.5%. Univariate analysis demonstrated that survival was affected by the following factors: (1) age: patients older than 55 had a better outcome (p = 0.03); (2) site of the tumour (p = 0.005): laryngeal tumours had the best survival; (3) clinical stage (p = 0.05). Histological grade did not influence the survival (p = 0.41). Patients having a tumour LI higher than 15.5% (mean + 1 S.D.) had a significantly lower survival than patients with lower tumour LI (p = 0.008). A multivariate analysis using the Cox model showed that clinical stage and LI kept their prognostic impact with regard to survival. Finally, survival after relapse was lower in patients with a high tumour LI. These results demonstrate that a high tumour proliferation rate is an additional factor influencing the disease outcome in head and neck carcinoma. Patients with bad prognosis defined by this parameter could be offered a more energetic treatment.     

Establishment and characterisation of a human clear cell sarcoma model in nude mice

We have established a new experimental model of human clear cell sarcoma, UM-CCS1, using serial subcutaneous transplantation of intact tumour tissue in nude mice. The heterotransplanted nude mouse tumours retained characteristic morphological features of the primary clear cell sarcoma. Immunohistochemical analysis showed the retained expression patterns of S-100 protein, melanoma-associated antigen HMB-45 and vimentin in the xenografts as compared to the primary tumour. DNA index showed low variations both between the xenografts in the same passage and between the serial passages. Cytogenetic analysis of the primary tumour and the xenografts showed the unbalanced translocation der(6)t(6;12)(p23;q13). Based on the combined genetic data a reasonable interpretation of our findings is that there was a complex chromosomal rearrangement resulting in a cytogenetically cryptic EWS-ATF1 fusion gene. Analysis of cell kinetics using in vivo incorporation of iododeoxyuridine and flow cytometry showed generally short potential doubling time (T(pot)) of the xenografts. Volume doubling time showed low variations without correlation with T(pot). The retained phenotypic and genotypic characteristics of the primary tumour and the morphological and structural stability over time makes the model suitable for studies on the tumour biology and treatment of clear cell sarcoma.     

Effectiveness of isolated liver perfusion with mitomycin C in the treatment of liver tumours of rat colorectal cancer

Dose limiting systemic toxicity prevents sufficient exploitation of the steep dose response relationship of most anticancer agents. In our rat liver tumour model (the CC531 colorectal carcinoma), isolated liver perfusion allows administration of higher doses of mitomycin C than hepatic artery infusion, while systemic toxicity remains minimal. To determine the temporal pattern of mitomycin C induced cytokinetic changes, we analysed flow cytometric DNA histograms of CC531 liver tumours from rats treated with high dose mitomycin C (3.2 mg kg-1) via hepatic artery infusion and sacrificed at different time intervals after treatment. Between 12 and 36 h after treatment, the fraction of cells in late S and G2/M phase had markedly increased. The effects of administration of the respective maximally tolerated doses of mitomycin C in isolated liver perfusion and via hepatic artery infusion on progression of tumour cells through the cell cycle and on gross tumour growth were compared. Isolated liver perfusion with mitomycin C resulted in a significant increase in the proportion of cells in mid and late S, and in some accumulation of cells in early S and G2/M phase at 24 and 48 h after treatment. In contrast, after hepatic artery infusion a significant increase of the fraction of cells in G2/M phase was observed at 24 h after treatment. Monitoring tumour growth after isolated liver perfusion five out of seven rats showed a complete tumour remission, while after hepatic artery infusion only a minimal growth delay was detected. This study demonstrates that isolated liver perfusion in the rat CC531 liver tumour model allows the administration of a well-tolerated dose of mitomycin C being high enough to induce a marked DNA synthesis inhibition and even complete tumour remission.     

Radiotherapeutic enhancement by razoxane

Razoxane blocks cell division in the G2/M phase of the cell generation cycle and appears to normalize tumour neovasculature development. These were the principal reasons for the examination and probably for the demonstration of the radiosensitizing activity of Rz. Specially intriguing has been the finding that radiation of primary tumour implants in animals treated with Rz has a powerful potentiating effect on the antimetastatic activity of the combination. This concept has not yet received any clinical examination. Clinical trials with radiotherapy and Rz have demonstrated the clearest beneficial effects in terms of response rates and maintained local control in soft tissue sarcomas and possibly in recurrent rectal and in bladder carcinomas. Interesting heightened activity has also been found in hepatic metastases from gastrointestinal tumours. Carcinomas of the cervix, bronchus and head and neck, however, have not shown any benefit from the combination compared with radiotherapy alone.     

Genistein Reinforces the Inhibitory Effect of Cisplatin on LiverCancer Recurrence and Metastasis after Curative Hepatectomy

Background: The high recurrence rate after hepatic resection in hepatocellular carcinoma (HCC) is a majorobstacle to improving prognosis. The objective of the present study was to explore the function of genistein, asoy-derived isoflavone, in enhancing the inhibitory effect of cisplatin on HCC cell proliferation and on tumorrecurrence and metastasis in nude mice after curative hepatectomy. Methods: Proliferation of human HCCcells (HCCLM3) was detected by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay.Synergistic effects of genistein and cisplatin were evaluated with the median-effect formula. Nude mice bearinghuman HCC xenografts underwent tumour resection (hepatectomy) 10 days post implantation, then receivedintraperitoneal administration of genistein or cisplatin alone or the combination of the two drugs. 33 days aftersurgery, recurrent tumours and pulmonary metastasis were evaluated individually. MMP-2 level in recurrenttumours was detected by immunohistochemistry and real-time PCR; MMP-2 expression in HCCLM3 was detectedby immunocytochemistry. Results: Genistein and cisplatin both suppressed the growth and proliferation ofHCCLM3 cells. The two drugs exhibited synergistic effects even at relatively low concentrations. In vivo, mice inthe combined genistein and cisplatin group had a smaller volume of liver recurrent tumors and fewer pulmonarymetastatic foci compared with single drug treated groups. Cisplatin upregulated the expression of MMP-2 in bothrecurrent tumours and HCCLM3, while genistein abolished cisplatin-induced MMP-2 expression. Conclusions:Genistein reinforced the inhibitory effect of cisplatin on HCC cell proliferation and tumour recurrence andmetastasis after curative hepatectomy in nude mice, possibly through mitigation of cisplatin-induced MMP-2upregulation.     

Growth-inhibitory and cell cycle-arresting properties of the rice bran constituent tricin in human-derived breast cancer cells in vitro and in nude mice in vivo

Tricin, a flavone found in rice bran, inhibits the growth of human-derived malignant MDA-MB-468 breast tumour cells at submicromolar concentrations. As part of the exploration of tricin as a potential cancer chemopreventive agent, we investigated the duration and cell cycle specificity of growth inhibition elicited by tricin in vitro and the effect of tricin on the development of MDA-MB-468 tumours grown in immune-compromised MF-1 mice in vivo. Preincubation of MDA-MB-468 cells with tricin (1-40 microM) for 72 h compromised cell growth after tricin removal, and such irreversibility was not observed in human breast-derived nonmalignant HBL-100 cells. Tricin (>/=5 microM) arrested MDA-MB-468 cells in the G2/M phase of the cell cycle without inducing apoptosis as adjudged by annexin V staining. In nude mice consumption of tricin with the diet (0.2%, w w(-1)) from 1 week prior to MDA-MB-468 cell implantation failed to impede tumour development. Steady-state levels of tricin in plasma, breast tumour tissue and intestinal mucosa, as measured by HPLC, were 0.13 microM and 0.11 and 63 nmol g(-1), respectively. Cells were exposed to tricin (0.11, 1.1 or 11 microM) in vitro for 72 h and then implanted into mice. The volume of tumours in animals bearing cells pre-exposed to 11 microM tricin was less than a third of that in mice with control cells, while tumours from cells incubated with 0.1 or 1.1 microM tricin were indistinguishable from controls. These results suggest that the potent breast tumour cell growth-inhibitory activity of tricin in vitro does not directly translate into activity in the nude mouse bearing the MDA MB-468 tumour. While the results do not support the notion that tricin is a promising candidate for breast cancer chemoprevention, its high levels in the gastrointestinal tract after dietary intake render exploration of its ability to prevent colorectal carcinogenesis propitious.