In vitro and in vivo antitumor efficacy of docetaxel and sorafenib combination in human pancreatic cancer cells.

The response of pancreatic cancer to treatments remains unsatisfactory, highlighting the need for more effective therapeutic regimens. Sorafenib, an orally available multikinase inhibitor, is active against different tumors, including pancreatic cancer. We studied the antitumor efficacy of sorafenib in combination with different antitumor drugs currently used in clinical practice in in vitro and in vivo experimental models of human pancreatic cancer. The cytotoxic effect of sorafenib and conventional antitumor drug combinations was evaluated in vitro in human pancreatic cancer cell lines and the efficacy of the most active combination was tested on tumor-bearing mice. Flow cytometric, Western blot and immunohistochemistry analyses were performed to investigate the mechanisms involved in the activity of single drugs and in their interaction when used in combination. Sorafenib showed a strong sequence-dependent synergistic interaction in vitro with docetaxel, which was highly dependent on the drug sequence employed. In vivo, human pancreatic cancer-xenografted mice treated with docetaxel followed by sorafenib reduced and delayed tumor growth, with complete tumor regression observed in half of the mice. This marked antitumor effect resulted in an overall increase in mouse survival of about 70% and in a complete cure in 3 of the 8 treated mice. The strong activity was also accompanied by marked apoptosis induction, inhibition of tumor angiogenesis and downregulation of ERK signalling. Our results show that the docetaxel and sorafenib combination exerts high therapeutic efficacy in experimental models of human pancreatic cancer, indicating a promising antitumor strategy for clinical use.

p53 nuclear accumulation and multiploidy are adverse prognostic factors in surgically resected stage II colorectal cancers independent of fluorouracil-based adjuvant therapy.

To identify the prognostically highest risk patients, DNA content and p53 nuclear or cytoplasmic accumulation, evaluated by monoclonal antibody DO7 and polyclonal antibody CM1, were determined in 94 surgically resected stage II (Dukes B2) colorectal cancers, treated or not with adjuvant 5-fluorouracil-based chemotherapy. Sixty-one (65%) of the tumors were aneuploid, 16 (17%) of which had a multiploid DNA content; 50 (53%) displayed DO7 nuclear p53 accumulation, and 44 (47%) showed cytoplasmic CM1 positivity. In multivariate analysis, only multiploidy and p53 nuclear positivity emerged as independent prognostic indicators of a poorer outcome. Positivity for p53 was associated with shorter survival in 5-fluorouracil-treated and untreated patients. Therefore, in patients with Dukes B2 colorectal cancer, a biologic profile based on the combined evaluation of DNA multiploidy and p53 status can provide valuable prognostic information, identifying patients to be enrolled in alternative, more aggressive therapeutic trials.

The application of hyperthermia in regional chemotherapy.

To evaluate the role of hyperthermia combined with chemotherapy in the loco-regional treatment of tumors, a retrospective analysis was done with 228 limb melanoma patients treated with hyperthermic antiblastic perfusion (HAP). A series of treatment- and tumor-related prognostic factors was analyzed to establish their influence on tumor response, loco-regional control, and survival. Concerning tumor response, the logistic model showed that the number of lesions and the minimal tumor temperature (min T) maintained their individual predictive values (P < 0.000001 and P = 0.04, respectively). For loco-regional control, only the number of lesions had a significant predictive value. No direct correlation was found between the treatment-related variables and loco-regional control. However, the 5-year survival rate was significantly higher for patients who achieved a complete response (CR) (51.5%, P = 0.0033) as compared to those who did not (33.3%), providing indirect evidence of the role of the treatment. Multivariate analysis showed that both disease-free and overall survival are strongly influenced by numerous clinical variables and the min T always maintained its significance. When analyzing the subgroup of 119 patients evaluable for tumor response, the Cox model selected the tumor response as the dominant factor for both disease-free and overall survival. These data seem to demonstrate that the optimization of treatment parameters is crucial in determining the CR rate, which, in turn, positively affects the disease outcome. HAP is the treatment of choice for recurrent limb melanoma, and hyperthermia plays an important role in exploiting the efficacy of this technique.

Modulation of the effects of the hyperthermic treatment by N-methylformamide on a human melanoma cell line.

The modulatory activity of the polar solvent N-methylformamide (NMF) on the effects of hyperhermic treatment was investigated on a human melanoma cell line (M14). Cells treated with NMF alone (1% for 20 h), hyperthermia (Hyp) alone (42.5 degrees C for 2 h) and with the two different sequences of treatment (NMF-->Hyp and Hyp-->NMF) were analysed by scanning electron microscopy and fluorescence microscopy. Moreover, their clonogenic efficiency and adherence properties were assessed. The results obtained can be summarized as follows. (a) The sequence Hyp-->NMF appeared to be more cytotoxic than the reverse sequence or NMF and Hyp given alone. (b) Heat induced cell swelling and detachment from the substrate. The pretreatment with the polar solvent was capable of preventing such alterations. (c) Fluorescence microscopy revealed remarkable changes induced by hyperthermia on actin network, vimentin distribution and vinculin expression. NMF administration proved to be capable of modulating these changes. In particular, the actin and vimentin networks showed a quite normal arrangement in NMF-->Hyp treated cells and very altered patterns in cells treated with the reverse sequence. Concerning the effects on the adhesion plaques, revealed by vinculin labeling, a considerable increase in the expression of these structures was observed after NMF treatment. (d) A remarkable increase of the attachment to collagen I and laminin molecules was revealed in NMF treated cells, whereas heat exposure reduced the number of adherent cells. Considered all together, the results obtained indicate that the administration of NMF after hyperthermia enhances the cytotoxic effect and modifies cell adherence properties, responsible for dissemination and metastasis.

Antiapoptotic effect of c-fes protooncogene during granulocytic differentiation.

The c-fes protooncogene is expressed at high levels in the terminal stages of granulocytic differentiation. Its product, p92c-fes, exhibits a tyrosine-kinase activity and is involved in the cellular response to GM-CSF, but its role is not yet clarified. To study this problem, the c-fes protooncogene expression has been inhibited in HL60 cells and in fresh leukemic blast cells of Acute Promyelocytic Leukemia (APL) induced to differentiate with All-Trans-Retinoic Acid (ATRA). Inhibition of c-fes function was obtained by treatment of the cells with a specific antisense oligomer complementary to the 5' region of the c-fes mRNA. It was observed that the cells, rather then differentiate to granulocytes, underwent premature cell death showing the morphological and molecular characteristics of apoptosis. Superimposable results are obtained on blast cells from APL. It is possible to conclude that the loss of cell viability that occurs during the in vitro differentiation of myeloid cells, after the complete inhibition of c-fes expression and treatment with ATRA, is due to activation of programmed cell death rather than an accelerated differentiation. Our data suggest that the c-fes product is essential for the survival of myeloid cells during differentiation.

Characterisation of a human glioblastoma cell line (LI) expressing hypothalamic and pituitary hormones.

The human glioblastoma cell line LI showed morphological features typical of its neuroectodermal origin. Cells were positive by immunofluorescence to GFAP, MHC class II, and L1 determinants. Cytogenetic analysis showed the presence of a modal chromosome number of 63, ranging from 58 to 69 chromosomes (DNA index was 1.6). Northern blot analysis demonstrated the presence of mRNA transcripts specific for transglutaminase C (type II or "tissue"), growth-hormone releasing-hormone (GHRH), insulin-like growth factor II (IGF-II), and proopiomelanocortin (POMC). The GHRH mRNA was present in two different sizes, one similar to the normal hypothalamic species of 0.75 kb, whilst the second species was a large transcript of approximately 10 kb size. Treatment with 5 microM retinoic acid or 5 mM alpha-difluoromethylornithine for 5 days sharply reduced the growth rate and also induced modulation of the ultrastructure and antigenic profile. This cell line may be useful to study glial differentiation and the relationship of GHRH, IGF-II and POMC expression with differentiation in neuroectodermal tumours.

Enhancement of hyperthermic damage on M14 melanoma cells by liposome pretreatment.

Exponentially growing human melanoma cells (M14 cell line) were pretreated with various amounts of dipalmitoylphosphatidylcholine-containing multilamellar liposomes and then exposed to heat treatment (42.5 degrees C). Cell damage produced by the treatments, given separately or in combination, was evaluated in terms of cell survival. Our results demonstrate that the cell survival at 37 degrees C was not affected by liposome concentrations up to 1000 nmol of phospholipid/2.5 x 10(6) cells, while liposome treatment of cells before heat exposure determined a marked damaging effect even at 100 nmol of phospholipid/2.5 x 10(6) cells. The mechanisms of liposome-cell interaction have been investigated by electron microscopy or by electron spin resonance measurements of spin-labeled membranes of intact cells. Evidence has been obtained that liposomal lipids are either taken up by M14 cells or become incorporated in the cell membrane. The present data suggest the possibility that liposome treatments per se could be of potential value as a therapeutic approach, by increasing the effect of heat therapy.

Effect of sequential application of hyperthermia and chemotherapy on the survival of a thermoresistant human melanoma cell line.

The human melanoma cell line M14 has been proven in previous experiments to be much less sensitive to the action of heat (42 degrees C, 60 min) than other melanoma lines. In the present study, we have investigated the possibility of increasing the effect of heat by means of drug treatment. Thermochemotherapy was applied to exponentially growing cells according to different schedulings, and was analyzed in its efficacy by measuring the impairment of the cellular colony-forming ability. Findings of the present study point out that: (a) appropriate sequencing between hyperthermia and cis-diamminedichloroplatinum II(DDP), melphalan (L-Pam), 1,2,4-dichlorobenzyl-1H-indazol-3-carboxylic acid (LNA) or 5-3,3-dimethyl-1-triazeno-imidazole-4-carboxamide (DTIC) strongly influences the cytotoxic effect of the two agents; and (b) the optimal combination appears to be the simultaneous application of heat and drugs. However, as far as thermochemotherapy with DDP is concerned, a synergistic effect may also be achieved when hyperthermia precedes DDP.

Kinetic and survival response of the M14 cell line to lonidamine associated with adriamycin or hyperthermia.

Lonidamine (LND), an indazole-carboxylic acid derivative, was delivered alone and together with adriamycin (ADM) or hyperthermia to the human melanoma cell line M14, and cell survival was assessed. Cell cycle-specific effects were investigated by analyzing sequences of DNA content histograms by means of a suitable mathematical procedure. LND delivered for 1 h at a dose of 50 micrograms/ml did not affect proliferation and survival of the cells. Exposure of the cells for 1 h to ADM (1.0 microgram/ml) followed by LND for 1 h (50 micrograms/ml) produced the highest effect on the survival. Kinetic parameters were affected by the combined treatment slightly more than by ADM exposure alone. Simultaneous delivery of LND (50 micrograms/ml) with hyperthermia (42 degrees C, 1 h) reduced the survival and enhanced the block of cells in the G2M phase, as compared with the heat treatment alone. The effect of the treatments on cell survival appeared to be related to the perturbation of the G2M phase of the cycle.