Gianfranco Canti

Bcl-2 phosphorylation and apoptosis activated by damaged microtubules require mTOR and are regulated by Akt

The serine/threonine kinase mTOR, the major sensor of cell growth along the PI3K/Akt pathway, can be activated by agents acting on microtubules. Damaged microtubules induce phosphorylation of the Bcl-2 protein and lower the threshold of programmed cell death, both of which are inhibited by rapamycin. In HEK293 cells expressing Akt mutants, the level of Bcl-2 phosphorylation and the threshold of apoptosis induced by taxol or by nocodazole are significantly modified. In cells expressing dominant-negative Akt (DN-Akt), Bcl-2 phosphorylation and p70S6KThr421/Ser424 phosphorylation induced by taxol or nocodazole were significantly enhanced as compared to cells expressing constitutively active Akt (CA-Akt) and inhibited by rapamycin. Moreover, DN-Akt cells were more sensitive to antitubule agents than CA-Akt cells. In nocodazole-treated HEK293 cells sorted according to cell cycle, the p70S6KThr421/Ser424 phosphorylation was associated to the G2/M fraction. More relevant, nocodazole inhibited, in a dose–response manner, mTOR phosphorylation at Ser2448. This activity, potentiated in DN-Akt cells, was not detectable in CA-Akt cells. Our results suggest that death signals originating from damaged microtubules in G2/M can compete with G1 survival pathways at the level of mTOR. These findings have implications for cancer therapy and drug resistance.

Preliminary evaluation of two fluorescence imaging methods for the detection and the delineation of basal cell carcinomas of the skin

Fluorescence techniques can provide powerful noninvasive means for medical diagnosis, based on the detection of either endogenous or exogenous fluorophores. The fluorescence of δ‐aminolevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) has already shown promise for the diagnosis of tumors. The aim of the study was to investigate the localization of skin tumors after the topical application of ALA, by detecting the PpIX fluorescence either in the spectral or in the time domain.

Antitumor efficacy of the combination of photodynamic therapy and chemotherapy in murine tumors

Photodynamic therapy (PDT) is based on the administration of tumor-localizing photosensitizers followed by light exposure of the tumor mass. The photocytotoxic effects are mainly caused by the generation of singlet oxygen. Recently, PDT has been proposed for use in combination with anticancer chemotherapy with a view to exploiting any additive antitumor effect. We investigated the effect of PDT with photoactivated aluminum disulfonated phthalocyanine (AlS2Pc) combined with the antiblastic drugs Adriamycin (ADR) and cisplatinum (CDDP) on murine tumors. Mice bearing L1210 leukemia and P388 lymphoma were treated with ADR or CDDP and subsequently treated with PDT. Low chemotherapy doses were ineffective, but the combination of antiblastic drugs + PDT had a significantly additive antitumor effect. In conclusion, with this combined therapy we were able to greatly reduce the effective doses of antiblastic drugs, thus lowering their toxic effects on normal host tissues.

Low doses of cisplatin or gemcitabine plus Photofrin/photodynamic therapy : Disjointed cell cycle phase-related activity accounts for synergistic outcome in metastatic non-small cell lung cancer cells (H1299)

We compared the effects of monotherapy (photodynamic therapy or chemotherapy) versus combination therapy (photodynamic therapy plus a specific drug) on the non–small cell lung cancer cell line H1299. Our aim was to evaluate whether the additive/synergistic effects of combination treatment were such that the cytostatic dose could be reduced without affecting treatment efficacy. Photodynamic therapy was done by irradiating Photofrin-preloaded H1299 p53/p16-null cells with a halogen lamp equipped with a bandpass filter. The cytotoxic drugs used were cis-diammine-dichloroplatinum [II] (CDDP or cisplatin) and 2′,2′-difluoro-2′-deoxycytidine (gemcitabine). Various treatment combinations yielded therapeutic effects (trypan blue dye exclusion test) ranging from additive to clearly synergistic, the most effective being a combination of photodynamic therapy and CDDP. To gain insight into the cellular response mechanisms underlying favorable outcomes, we analyzed the H1299 cell cycle profiles and the expression patterns of several key proteins after monotherapy. In our conditions, we found that photodynamic therapy with Photofrin targeted G0-G1 cells, thereby causing cells to accumulate in S phase. In contrast, low-dose CDDP killed cells in S phase, thereby causing an accumulation of G0-G1 cells (and increased p21 expression). Like photodynamic therapy, low-dose gemcitabine targeted G0-G1 cells, which caused a massive accumulation of cells in S phase (and increased cyclin A expression). Although we observed therapeutic reinforcement with both drugs and photodynamic therapy, reinforcement was more pronounced when the drug (CDDP) and photodynamic therapy exert disjointed phase-related cytotoxic activity. Thus, if photodynamic therapy is appropriately tuned, the dose of the cytostatic drug can be reduced without compromising the therapeutic response. [Mol Cancer Ther 2006;5(3):776–85]

In vivo assimilation of low density lipoproteins by a fibrosarcoma tumour line in mice

A tumour line inoculated in mice showed high affinity binding for lipoproteins in vitro. Studies in vivo demonstrated that the assimilation of human low density lipoprotein (LDL) by the tumour was very high. Both receptor and non-receptor mediated catabolism of the lipoprotein by the tumour increased as compared to other tissues known to be sites of lipoprotein catabolism (liver, spleen etc.). These findings suggest that lipoproteins may be useful markers for tumours as well as carriers for cytotoxic drugs to target tissues in vivo.

TIME‐GATED FLUORESCENCE IMAGING FOR THE DIAGNOSIS OF TUMORS IN A MURINE MODEL

A system for time‐gated fluorescence imaging was used to perform measurements on tumor‐bearing mice treated with hematoporphyrin derivative (HpD). The aim of the study was to define the potential of this technique in the diagnosis of tumors by taking advantage of the long fluorescence lifetime of the exogenous dye with respect to the decay times of the natural fluorescence. After the administration of three different drug doses (5, 10 and 25 mg/kg body weight), fluorescence images were acquired at various uptake times (from 2 h to 10 d), to determine the best instrumental conditions and experimental procedure for the detection of tumors in the murine model considered. The optimal fluorescence contrast between the tumor area and the surrounding healthy tissue was found at 12 h after the administration of either 5 or 10 mg/kg HpD and was anticipated at 8 h for the highest drug dose. In this optimum condition, the tumor region could be identified even after the injection of 5 mg/kg HpD. A better fluorescence contrast was always obtained in 15 ns‐delayed images with respect to synchronous ones.

FLUORESCENCE LIFETIME IMAGING OF EXPERIMENTAL TUMORS IN HEMATOPORPHYRIN DERIVATIVE-SENSITIZED MICE

Tumor detection has been carried out in mice sensitized with hematoporphyrin derivative (HpD) by measuring the spatial distribution of the fluorescence lifetime of the exogenous compound. This result has been achieved using a time‐gated video camera and a suitable mathematical processing that led to the so‐called “lifetime images.” Extensive experimental tests have been performed on mice bearing the MS‐2 fibrosarcoma or the L1210 leukemia. Lifetime images of mice show that the fluorescence decay of HpD is appreciably slower in the tumor than in healthy tissues nearby, allowing a reliable detection of the neoplasia. The lengthening of the lifetime in tumors depends little on the drug dose, which in our experiments could be lowered down to 0.1 mg/kg body weight, still allowing a definite tumor detection. In order to ascertain the results achieved with the imaging apparatus, high‐resolution spectroscopy, based on a time‐correlated single photon counting system, has also been performed to measure the fluorescence lifetime of the drug inside the tumor and outside. The outcomes obtained with two techniques are in good agreement.

Photodynamic therapy and the immune system in experimental oncology

Photodynamic therapy (PDT), a newly established treatment for solid tumors, involves the systemic administration of a tumor localizing photosensitizer that is only activated when exposed to light of appropriate wavelength. Photoactivation of the photosensitizer in the presence of oxygen results in the generation of highly cytotoxic molecular species. The PDTmediated antitumor effect is oxygen dependent and is the consequence of direct cytotoxicity and an antivascular effect which impairs blood supply to the area. Furthermore, the development of a generalised inflammatory state in the treatment region and immunological mechanism may contribute to tumor regression. The most widely studied PDT drugs both in experimental and clinical trials have been hematoporphyrin derivative and Photofrin (a complex mixture of monomeric and oligomeric porphyrins). While encouraging results have been obtained with these drugs, their documented limitations have led to the search for second generation photosensitizers in the hope of increasing the efficacy of the treatment (clearly definable molecular structure, activation at longer, more penetrating wavelengths of light and a more rapid clearance from the subject). Second generation photosensitizers that are currently under clinical evaluation include tin etiopurpurin dichloride (SnET2), lutetium texaphyrin, meso-tetrahydroxyphenylchlorin (m-THPC), phthalocyanines and benzoporphyrin derivatives, monoacid ring A (BPD-MA, Verteporfin) and ALA (5-aminolevulinic acid).

Rapamycin increases the cellular concentration of the BCL-2 protein and exerts an anti-apoptotic effect

The immunosuppressant rapamycin, an immunophilin-binding antibiotic, has been studied in follicular B-cell lymphoma lines that express the highest level of the BCL-2 protein. The growth rate of human follicular B-cell lymphoma lines was slowed more efficiently than that of other human B-cell lines or non-B-cell lines. This effect was dependent on the arrest of cells in the G(1) phase; the number of apoptotic cells was not increased. Rapamycin inhibited apoptosis or caspase activation induced by cytotoxic drugs, whereas caspase activation by doxorubicin was not inhibited. The increase in the cellular concentration of BCL-2 protein was related to its concentration in the steady state and was unrelated to the amount of bcl-2 mRNA. The increase of BCL-2 level in the cells rather than its level in the steady state may be important for drug resistance. The biochemical target of rapamycin, the mTOR kinase, may be a candidate sensitising agent for chemotherapy. This effect of rapamycin shows that G(1) arrest and protection from apoptosis are combined events susceptible to regulation by pharmacological means.

Fluorescence molecular tomography of an animal model using structured light rotating view acquisition

Abstract. In recent years, an increasing effort has been devoted to the optimization of acquisition and reconstruction schemes for fluorescence molecular tomography (FMT). In particular, wide-field structured illumination and compression of the measured images have enabled significant reduction of the data set and, consequently, a decrease in both acquisition and processing times. FMT based on this concept has been recently demonstrated on a cylindrical phantom with a rotating-view scheme that significantly increases the reconstruction quality. In this work, we generalize the rotating-view scheme to arbitrary geometries and experimentally demonstrate its applicability to murine models. To the best of our knowledge this is the first time that FMT based on a rotating-view scheme with structured illumination and image compression has been applied to animals.