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The relationship between modification of energy metabolism and extent of drug resistance was investigated in two sublines (LoVoDX and LoVoDX10) from human LoVo colon carcinoma cells that exhibit different degrees of resistance to doxorubicin. Results indicated that the extent of alteration in energy metabolism strictly correlated with degree of resistance. In LoVoDX cells, only 14CO2 production was enhanced, whereas in the more resistant LoVoDX10 cells, both 14CO2 and aerobic lactate production were stimulated. The basal and glucose-supported efflux rate and the amount of drug extruded by LoVoDX10 cells were significantly higher than in the resistant LoVoDX cells. Because the expression of surface P-170 glycoprotein was similar in both cell lines, this phenomenon was attributed to increased efflux pump activity resulting from greater ATP availability. Inhibition of 14CO2 production, aerobic glycolysis, and clonogenic activity by lonidamine (LND) increased with enhancement of the energy metabolism. Moreover, LND, by affecting energy-yielding processes, reduced intracellular ATP content, lowered the energy supply to the ATP-driven efflux pump, and inhibited, almost completely, doxorubicin extrusion by resistant LoVo cells. These findings strongly suggest that LND, currently used in tumor therapy, reduces drug resistance by restoring the capacity to accumulate and retain drug of cells with the MDR phenotype that overexpress P-170.

Overexpression of gp185(erbB-2) has been associated with reduced survival in breast-cancer patients. Our earlier results, now confirmed in a larger cohort of patients (798), evidenced that the HLA-A2 allele may participate in the modulation of the erbB-2 tumor phenotype in vivo. In the present study, we evaluated other clinico-biopathologic parameters possibly involved in the host immune response against erbB-2. Localization of the CD3(+) T-cell infiltrate was taken into consideration in 705 primary breast tumors, and expression of HLA-class-I and HLA-A2 antigens was evaluated in a subgroup of 170 frozen primary tumors of HLA-A2-positive patients. The presence or the absence of HLA-class-I and HLA-A2 antigens in primary tumors did not correlate with erbB-2 expression. However, HLA-A2-positive tumors preferentially showed intratumoral lymphocyte localization, whereas the lesions displaying undetectable HLA-class-I expression showed peritumoral CD3(+) T-cell localization. Taking into account erbB-2 immunoreactivity, we found that the relationship between HLA-A2 expression and intratumoral CD3(+) T-lymphocyte localization is significant only in the erbB-2 negative subset, whereas the relationship between lack of HLA-class-I expression and peritumoral CD3(+) T-lymphocyte localization is significant only in the erbB-2-positive subset. These data provide novel in vivo evidence of the possible contribution of the host immune system to control of erbB-2 oncogene overexpression in breast cancer. Int. J. Cancer (Pred. Oncol.) 84:598-603, 1999., (Copyright 1999 Wiley-Liss, Inc.)

The effect of hyperthermia and bupivacaine, alone and in combination, on the clonogenic activity of a human melanoma cell line was investigated. The time-temperature relationship of exponentially growing cells was defined in the range of 41-45 degrees C. All the survival curves were exponential and the Arrhenius plot was linear over the temperature range tested. The survival curve of bupivacaine-treated cells was also exponential after an initial shoulder. Bupivacaine affected cell survival mainly through an ATP depletion because of deep alterations of mitochondria, essentially due to changes in the physical state of membrane lipids. The analysis of the interaction between hyperthermia and bupivacaine, performed with an isobolar method, demonstrated a synergism of response at all combinations tested, but only with simultaneous exposure. Such a response did not depend on an impairment of the energy-yielding processes, but may be ascribed to combined effects of both agents on cell structure and function. The hyperthermic enhancement achieved by low bupivacaine concentrations allowed to achieve a preestablished cell killing with a reduced exposure time (e.g., 50 min) and with a temperature (42 degrees C) generally accepted as clinically achievable. Therefore, a combined modality in which local treatment with bupivacaine was coupled to local heating could result in high local damage with reduced systemic complications.

Small peptides, 8-10 amino acids long, derived from degradation of cytoplasmic proteins by a proteasome-proteinase complex, are usually presented and recognized by CD8+ cytolytic T lymphocytes (CTLs) associated with major histocompatibility complex (MHC) class I molecules. Recently synthetic peptides were used for the in vitro induction of tumor-specific CTLs, offering another strategy in the study of the immune-response repertoire and providing a new tool in cancer vaccination and immunotherapy. Peptides derived from otherwise normal proteins, overexpressed in many tumors as products of the protooncogene, may represent a target for an immune response. This is the case of HER-2/neu gene (also known as ErbB-2), encoding a cysteine-rich glycoprotein transmembrane receptor with tyrosine kinase activity (gp185neu). Recent data, demonstrating that HLA-A2.1-related peptides are able to stimulate in vitro CD8+ lymphocytes, Prompted us to study the binding to HLA-A2.1 molecules of several gp185 synthetic peptides containing a cystein residue and to define the relevance of this amino acid residue in the reduced or oxidated form of the sulfhydryl group. We found that monomers and their homodimers, linked by a disulfide bridge, bind to HLA-A2.1 molecules with overlapping affinity. These results suggest that additional amino acids of the nonapeptide do not prevent the binding and the HLA refolding through chemical or sterical interactions. This might be of particular relevance for the in vivo processing of cysteine-rich proteins. Because ErbB-2 molecules, as tumor-differentiation antigens in melanoma, are cysteine-rich molecules, it may be relevant to evaluate the possible role of the cystine residues interacting with the T-cell receptor. The recognition of these heterodimers by CD8+ lymphocytes will require functional in vivo studies.