P53 and PTEN expression contribute to the inhibition of EGFR downstream signaling pathway by cetuximab.

Cetuximab (Erbitux) is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody whose activity is related to the inhibition of EGFR downstream signaling pathways. P53 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been reported to control the functionality of PI3K/AKT signaling. In this study we evaluated whether reintroducing P53 using non-viral gene transfer enhances PTEN-mediated inhibition of PI3K/AKT signaling by cetuximab in PC3 prostate adenocarcinoma cell line bearing p53 and pten mutations. Signaling phosphoproteins expression was analyzed using Bio-Plex phosphoprotein array and western blot. Apoptosis induction was evaluated from BAX expression, caspase-3 activation and DNA fragmentation analyses. The results presented show that p53 and pten gene transfer additionally mediated cell growth inhibition and apoptosis induction by restoral of signaling functionality, which enabled the control of PI3K/AKT and MAPKinase signaling pathways by cetuximab in PC3 cells. These results highlight the interest of the analysis of signaling phosphoproteins expression as molecular predictive markers for response to cetuximab and show that p53 and pten mutations could be key determinants of cell response to cetuximab through the functional impact of these mutations on cell signaling.

Oncoprotein expression of E6 and E7 does not prevent 5-fluorouracil (5FU) mediated G1/S arrest and apoptosis in 5FU resistant carcinoma cell lines.

5-Fluorouracil (5FU) exposure can lead to both G1/S arrest and apoptosis induction which are dependent of P53 induction. The human papilloma virus oncoproteins (HPV), E6 and E7, inactivate respectively P53 and Rb. P53 degradation by E6 protein, leads to lack of G1/S arrest after genotoxic stress. Overexpression of E7 protein prevents P53-induced G1/S arrest following DNA damage. However, few studies have described 5FU effect and efficacy on cancer cell lines presenting HPV 18 positive status. KB cell line and KB3 subline presented wild-type P53 status and difference in 5FU sensitivity. During 5FU exposure, P53 gene and protein expression was increased in both cell lines. E6 and E7 mRNA and protein expression was decreased in KB and KB3. P53 and E6 protein expressions were inversely correlated. 5FU exposure, induced a G1/S arrest which can be maintained or intensified by P53 via P21 induction expression. 5FU exposure has led to apoptosis induction related to P53 induction. In the present study, 5FU exposure was shown to induce G1/S arrest and apoptosis by P53-dependent molecular pathway, in HPV 18 positive cells.

Radiation could induce p53-independent and cell cycle--unrelated apoptosis in 5-fluorouracil radiosensitized head and neck carcinoma cells.

The effect of chemoresistance induction in radiosensitivity and cellular behavior after irradiation remains misunderstood. This study was designed to understand the relationship between radiation-induced cell cycle arrest, apoptosis, and radiosensitivity in KB cell line and KB3 subline selected after 5-fluorouracil (5FU) exposure. Exposure of KB cells to 5FU led to an increase in radiosensitivity. G2/M cell cycle arrest was observed in the two cell lines after irradiation. The radioresistant KB cell line reached the maximum arrest two hours before KB3. The cellular exit from this arrest was found to be related to the wild type p53 protein expression induction. After irradiation, only KB3 cell line underwent apoptosis. This apoptosis induction seemed to be independent of G2/M arrest exit, which was carried out later. The difference in radiosensitivity between KB and KB3 subline may result therefore from both a difference in apoptosis induction and a difference in G2/M arrest maximum duration. Moreover, 5FU exposure has led to an increase in constitutive p53 protein expression, which may be associated with an increase in basal apoptosis cell fraction. Given the existing correlation between radiosensitivity and the percentage of basal apoptosis, the constitutive p53 protein expression may be related to intrinsic radiosensitivity in our cellular model.

Modulation of daunorubicin cellular resistance by combination of P-glycoprotein blockers acting on drug efflux and intracellular drug sequestration in Golgi vesicles.

BACKGROUND: S9788 and PSC833 were developped as P-glycoprotein (Pgp) blockers and found to act additionally on daunorubicin subcellular distribution, involving different putative targets. On this basis, combinations of S9788 and PSC833 were evaluated in Pgp-expressing MCF7(DXR) cells in which we recently demonstrated that daunorubicin was sequestered in Golgi vesicles (Bour-Dill et al.: Cytometry, 39: 16-25, 2000). METHODS: Combinations of S9788 and PSC833 consisted in complementary fractions of iso-effective concentrations (IEC) leading to 90% (IEC90) and median (IEC50) reversion of daunorubicin resistance. Resistance modulation was assessed using cytotoxicity assays, flow cytometry determination of intracellular daunorubicin, and fluorescence microscopy analysis of daunorubicin subcellular distribution. RESULTS: Individually, both S9788 and PSC833 were found to be very potent with IEC90 of 5 and 15 micromol/l, and IEC50 of 0.1 and 0.2 micromol/l, respectively, for S9788 and PSC833. When combined, synergistic cytotoxicity was observed for both IEC90 and IEC50 combinations while intracellular daunorubicin fluorescence was only synergistically increased for IEC90 combinations. For IEC50 combinations, no increase in intracellular fluorescence was observed, and fluorescence microscopy examination of the cells suggested that daunorubicin sequestration in Golgi vesicles could be modulated at concentrations that do not significantly increase daunorubicin cellular concentration. Using immunofluorescence and reverse transcription-polymerase chain reaction analyses, multidrug resistance-associated protein, major vault lung-resistance protein, and anthracycline-resistance associated protein were not found to be implicated. CONCLUSIONS: Synergistic combinations of S9788 and PSC833 might offer alternative ways to decrease the toxicity generated by high-dose Pgp-blockers without altering the efficacy of the resistance modulation.

Modulation of daunorubicin intracellular accumulation in P-glycoprotein expressing MCF-7 human breast adenocarcinoma cells by thermosensitive-liposome encapsulation and hyperthermia.

Intracellular accumulation of free or thermosensitive liposome-encapsulated daunorubicin (TLED) at 37 or 43 degrees C, was evaluated using flow cytometry in chemosensitive and P-glycoprotein expressing MCF-7 human breast adenocarcinoma cells. At 37 degrees C, liposome-encapsulation significantly increased intracellular daunorubicin accumulation (IDA) in resistant cells (P=0.005) and that effect was statistically comparable to that achieved in adding 15 micromol/l verapamil to free daunorubicin (DNR). Combining TLED and verapamil further enhanced significantly (P=0.004) this effect as compared to TLED alone. However, none of these treatments restored IDA to the level achieved in sensitive cells. Hyperthermia significantly increased IDA in the sensitive cell (P<0.05), whenever free-DNR or TLED was used, but had no significant effect in the resistant cells, suggesting that P-glycoprotein could efflux the additional drug uptaken in hyperthermic conditions. In all these experiments combining the use of a modulator (verapamil or TLED) and hyperthermia, IDA was statistically comparable to that achieved with free-DNR in sensitive cells at 37 degrees C, but still remained lower than the IDA in sensitive cells at 43 degrees C (P<0.05). The results also showed that hyperthermia affected the labelling of P-glycoprotein by MRK16 monoclonal antibody.

Influence of S9788, a new modulator of multidrug resistance, on the cellular accumulation and subcellular distribution of daunorubicin in P-glycoprotein-expressing MCF7 human breast adenocarcinoma cells.

A triazinoaminopiperidine derivative synthesized as a modulator of multidrug resistance, S9788, was investigated in the human breast adenocarcinoma MCF7DXR cell line expressing P-glycoprotein. In addition to being less sensitive to daunorubicin, the resistant cell line showed dramatic alterations in the subcellular distribution of daunorubicin, as observed via fluorescence microscopy and quantified via tritiated daunorubicin nuclear distribution analysis. Compared to verapamil and cyclosporin A at 2 and 5 mumol/liter, S9788 proved to be more potent in restoring the cellular accumulation and the subcellular distribution of daunorubicin in the resistant cells. Significant activity of S9788 was observed at 2 mumol/liter, which is clinically achievable, and S9788 restored the nuclear distribution of the drug to the level observed in the parental sensitive cell line. Consequently, the restoration of the cytotoxicity of daunorubicin by S9788 was nearly complete (> 90%) at 2 mumol/liter, wheras cyclosporin A reached this level of activity at 5 mumol/liter, and verapamil was always less active at both concentrations. These results suggest that the modulation of multidrug resistance by S9788 is not only related to the enhancement of the cellular accumulation but also especially by the restoration of the subcellular distribution of the drugs to their nuclear sites of action.

Comparative evaluation of S9788, verapamil, and cyclosporine A in K562 human leukemia cell lines and in P-glycoprotein-expressing samples from patients with hematologic malignancies.

The activity of S9788, recently synthetized as a modulator of multidrug resistance (MDR), was compared with verapamil and cyclosporine A in normal sensitive and MDR K562 cell lines, then in samples from 33 patients with hematological malignancies, using flow cytometry with simultaneous detection of P-glycoprotein and determination of intracellular daunorubicin fluorescence. This technique was compared and correlated with a tritiated daunorubicin accumulation method. In K562 cell lines, S9788 exhibited a significantly higher reversing activity than verapamil and cyclosporine A, and allowed a complete restoration of the accumulation of daunorubicin when used at 5 mumol/L. In the clinical samples, the three compounds were evaluated at equimolar concentration (5 mumol/L) using concomitant exposure to daunorubicin and to the reversing agent. In P-glycoprotein-negative samples, no significant effect on intracellular daunorubicin fluorescence of any of the reversing agents was noted. In the 15 P-glycoprotein-positive samples, a significant increase in daunorubicin fluorescence, by at least one reversing agent, was seen in 10 cases, among which S9788 reversing activity was higher than that of the two other agents in seven cases. Complete reversal was only achieved in one case with S9788.

Antiproliferative activity of thermosensitive liposome-encapsulated doxorubicin combined with 43 degrees C hyperthermia in sensitive and multidrug-resistant MCF-7 cells.

Thermosensitive liposome-encapsulated doxorubicin (TLED) was compared to free doxorubicin, at 37 degrees C or combined with 43 degrees C hyperthermia, in sensitive and multidrug-resistant MCF-7 human tumour cells using clonogenic assays. In the resistant subline, TLED was found to partly circumvent multidrug resistance (MDR). The reversal was comparable to that obtained when verapamil was added to free doxorubicin. When hyperthermic treatment was applied, no difference in thermosensitivity was found between sensitive and resistant cells. The combination of hyperthermia with free doxorubicin did not reverse MDR. Hyperthermia and TLED yielded additive effects in the resistant cells while potentiation was observed in the sensitive cells. These results confirmed the usefulness of the liposome encapsulation of doxorubicin in reversing MDR. The possibility of obtaining additive cytotoxicity using TLED combined with hyperthermia may represent an alternative way of intensification of doxorubicin cytotoxicity concomitant with the circumvention of MDR without using MDR reversing agents, which often generate limiting toxic side-effects.

MTT assays allow quick and reliable measurement of the response of human tumour cells to photodynamic therapy.

MCF-7 and HT-29 cell lines were selected as a reliable model to examine the possible parameters affecting the sensitivity of tumour cells to photodynamic therapy (PDT) using a dye-laser at 630 nm. The chemical composition of haematoporphyrin derivative (HPD) was determined by high-performance liquid chromatography (HPLC) analysis and was in agreement with reported values. MTT assays were performed to assess the time-dependency of PDT and the influence of the output power and light fluence. The results showed a maximal cytotoxicity 48 h after photoirradiation. The output power (1 or 2 W) did not significantly affect the cytotoxicity when the fluence was constant (20 J/cm2). However, an increase in fluence (10-40 J/cm2) led to a significant enhancement of cytotoxicity until maximal values were reached (30-40 J/cm2). A further increase in fluence (50 J/cm2) proved to induce a fall-off in cytotoxicity related to the intense photobleaching of HPD.

Modulation by palmitoyl-carnitine of calcium activated, phospholipid-dependent protein kinase activity and inhibition of melanoma cell growth.

Phospholipid-sensitive, Ca++-dependent protein kinase activity was investigated in the cytosol of melanoma cells. A protein kinase system was partially purified, and enzyme activity was found to be modulated by palmitoyl-carnitine. In order to link the actions of palmitoyl-carnitine on phospholipid-sensitive protein kinase activity and the already reported role of protein kinase C in cell division, we studied the action of palmitoyl-carnitine on melanoma cell growth by measuring colony forming ability in a soft agar culture system. Palmitoyl-carnitine was found to inhibit cell growth in a dose-dependent manner. These findings suggest that palmitoyl-carnitine (or long-chain acylcarnitine), a naturally occurring metabolite, may play a key role in the onset of cell division. We suggest that the action of palmitoyl-carnitine on phospholipid-dependent protein kinase activity is in part related to the molecular events linking protein kinase C activity and the ionic events in the initiation of cell growth.