Chloroquine supplementation increases the cytotoxic effect of curcumin against Her2/neu overexpressing breast cancer cells in vitro and in vivo in nude mice while counteracts it in immune competent mice.

Autophagy is usually a pro-survival mechanism in cancer cells, especially in the course of chemotherapy, thus autophagy inhibition may enhance the chemotherapy-mediated anti-cancer effect. However, since autophagy is strongly involved in the immunogenicity of cell death by promoting ATP release, its inhibition may reduce the immune response against tumors, negatively influencing the overall outcome of chemotherapy. In this study, we evaluated the in vitro and in vivo anti-cancer effect of curcumin (CUR) against Her2/neu overexpressing breast cancer cells (TUBO) in the presence or in the absence of the autophagy inhibitor chloroquine (CQ). We found that TUBO cell death induced by CUR was increased in vitro by CQ and slightly in vivo in nude mice. Conversely, CQ counteracted the Cur cytotoxic effect in immune competent mice, as demonstrated by the lack of in vivo tumor regression and the reduction of overall mice survival as compared with CUR-treated mice. Immunohistochemistry analysis revealed the presence of a remarkable FoxP3 T cell infiltrate within the tumors in CUR/CQ treated mice and a reduction of T cytotoxic cells, as compared with single CUR treatment. These findings suggest that autophagy is important to elicit anti-tumor immune response and that autophagy inhibition by CQ reduces such response also by recruiting T regulatory (Treg) cells in the tumor microenvironment that may be pro-tumorigenic and might counteract CUR-mediated anti-cancer effects.

ZnCl2 sustains the adriamycin-induced cell death inhibited by high glucose.

Hyperglycemia, the condition of high blood glucose, is typical of diabetes and obesity and represents a significant clinical problem. The relationship between hyperglycemia and cancer risk has been established by several studies. Moreover, hyperglycemia has been shown to reduce cancer cell response to therapies, conferring resistance to drug-induced cell death. Therefore, counteracting the negative effects of hyperglycemia may positively improve the cancer cell death induced by chemotherapies. Recent studies showed that zinc supplementation may have beneficial effects on glycemic control. Here we aimed at evaluating whether ZnCl2 could counteract the high-glucose (HG) effects and consequently restore the drug-induced cancer cell death. At the molecular level we found that the HG-induced expression of genes known to be involved in chemoresistance (such as HIF-1α, GLUT1, and HK2 glycolytic genes, as well as NF-κB activity) was reduced by ZnCl2 treatment. In agreement, the adryamicin (ADR)-induced apoptotic cancer cell death was significantly impaired by HG and efficiently re-established by ZnCl2 cotreatment. Mechanistically, the ADR-induced c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) phosphorylation, inhibited by HG, was efficiently restored by ZnCl2. The JNK involvement in apoptotic cell death was assessed by the use of JNK dominant-negative expression vector that indeed impaired the ZnCl2 ability to restore drug-induced cell death in HG condition. Altogether, these findings indicate that ZnCl2 supplementation efficiently restored the drug-induced cancer cell death, inhibited by HG, by both sustaining JNK activation and counteracting the glycolytic pathway.

Targeting MKK3 as a novel anticancer strategy: molecular mechanisms and therapeutical implications.

Mitogen-activated protein kinase kinase 3 (MAP2K3, MKK3) is a member of the dual specificity protein kinase group that belongs to the MAP kinase kinase family. This kinase is activated by mitogenic or stress-inducing stimuli and participates in the MAP kinase-mediated signaling cascade, leading to cell proliferation and survival. Several studies highlighted a critical role for MKK3 in tumor progression and invasion, and we previously identified MKK3 as transcriptional target of mutant (mut) p53 to sustain cell proliferation and survival, thus rendering MKK3 a promising target for anticancer therapies. Here, we found that targeting MKK3 with RNA interference, in both wild-type (wt) and mutp53-carrying cells, induced endoplasmic reticulum stress and autophagy that, respectively, contributed to stabilize wtp53 and degrade mutp53. MKK3 depletion reduced cancer cell proliferation and viability, whereas no significant effects were observed in normal cellular context. Noteworthy, MKK3 depletion in combination with chemotherapeutic agents increased tumor cell response to the drugs, in both wtp53 and mutp53 cancer cells, as demonstrated by enhanced poly (ADP-ribose) polymerase cleavage and reduced clonogenic ability in vitro. In addition, MKK3 depletion reduced tumor growth and improved biological response to chemotherapeutic in vivo. The overall results indicate MKK3 as a novel promising molecular target for the development of more efficient anticancer treatments in both wtp53- and mutp53-carrying tumors.

Degradation of mutant p53H175 protein by Zn(II) through autophagy.

TP53, one of the most important oncosuppressors, is frequently mutated in cancer. Several p53 mutant proteins escape proteolytic degradation and are highly expressed in an aberrant conformation often acquiring pro-oncogenic activities that promote tumor progression and resistance to therapy. Therefore, it has been vastly proposed that reactivation of wild-type (wt) function(s) from mutant p53 (mutp53) may have therapeutic significance. We have previously reported that Zn(II) restores a folded conformation from mutp53 misfolding, rescuing wild-type (wt) p53/DNA-binding and transcription activities. However, whether Zn(II) affects mutp53 stability has never been investigated. Here we show that a novel Zn(II) compound induced mutp53 (R175H) protein degradation through autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins. Accordingly, pharmacological or genetic inhibition of autophagy prevented Zn(II)-mediated mutp53H175 degradation as well as the ability of the Zn(II) compound to restore wtp53 DNA-binding and transcription activity from this mutant. By contrast, inhibition of the proteasome failed to do so, suggesting that autophagy is the main route for p53H175 degradation. Mechanistically, Zn(II) restored the wtp53 ability to induce the expression of the p53 target gene DRAM (damage-regulated autophagy modulator), a key regulator of autophagy, leading to autophagic induction. Accordingly, inhibition of wtp53 transactivation by pifithrin-α (PFT-α) impaired both autophagy and mutp53H175 degradation induced by curcumin-based zinc compound (Zn(II)-curc). Viewed together, our results uncover a novel mechanism employed by Zn(II)-curc to reactivate mutp53H175, which involves, at least in part, induction of mutp53 degradation via wtp53-mediated autophagy.

HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma.

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-µ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.

Glucose restriction induces cell death in parental but not in homeodomain-interacting protein kinase 2-depleted RKO colon cancer cells: molecular mechanisms and implications for tumor therapy.

Tumor cell tolerance to nutrient deprivation can be an important factor for tumor progression, and may depend on deregulation of both oncogenes and oncosuppressor proteins. Homeodomain-interacting protein kinase 2 (HIPK2) is an oncosuppressor that, following its activation by several cellular stress, induces cancer cell death via p53-dependent or -independent pathways. Here, we used genetically matched human RKO colon cancer cells harboring wt-HIPK2 (HIPK2(+/+)) or stable HIPK2 siRNA interference (siHIPK2) to investigate in vitro whether HIPK2 influenced cell death in glucose restriction. We found that glucose starvation induced cell death, mainly due to c-Jun NH2-terminal kinase activation, in HIPK2(+/+)cells compared with siHIPK2 cells that did not die. (1)H-nuclear magnetic resonance quantitative metabolic analyses showed a marked glycolytic activation in siHIPK2 cells. However, treatment with glycolysis inhibitor 2-deoxy-D-glucose induced cell death only in HIPK2(+/+) cells but not in siHIPK2 cells. Similarly, siGlut-1 interference did not re-establish siHIPK2 cell death under glucose restriction, whereas marked cell death was reached only after zinc supplementation, a condition known to reactivate misfolded p53 and inhibit the pseudohypoxic phenotype in this setting. Further siHIPK2 cell death was reached with zinc in combination with autophagy inhibitor. We propose that the metabolic changes acquired by cells after HIPK2 silencing may contribute to induce resistance to cell death in glucose restriction condition, and therefore be directly relevant for tumor progression. Moreover, elimination of such a tolerance might serve as a new strategy for cancer therapy.

Regulation of p53 activity by HIPK2: molecular mechanisms and therapeutical implications in human cancer cells.

The p53 protein is the most studied tumor suppressor and the p53 pathway has been shown to mediate cellular stress responses that are disrupted when cancer develops. After DNA damage, p53 is activated as transcription factor to directly induce the expression of target genes involved in cell-cycle arrest, DNA repair, senescence and, importantly, apoptosis. Post-translational modifications of p53 are essential for the activation of p53 and for selection of target genes. The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating its N-terminal serine 46 (Ser46) and facilitating Lys382 acetylation at the C-terminus. HIPK2 is activated by numerous genotoxic agents and can be deregulated in tumors by several conditions including hypoxia. Recent findings suggest that HIPK2 active/inactive protein can affect p53 function in multiple and unexpected ways. This makes p53 as well as HIPK2 interesting targets for cancer therapy. Hence, understanding the role of HIPK2 as p53 activator may provide important insights in the process of tumor progression, and may also serve as the crucial point in the diagnostic and therapeutical aspects of cancer.

p53 can inhibit cell proliferation through caspase-mediated cleavage of ERK2/MAPK.

Stimulation of the Ras/MAPK cascade can either activate p53 and promote replicative senescence and apoptosis, or degrade p53 and promote cell survival. Here we show that p53 can directly counteract the Ras/MAPK signaling by inactivating ERK2/MAPK. This inactivation is due to a caspase cleavage of the ERK2 protein and contributes to p53-mediated growth arrest. We found that in Ras-transformed cells, growth arrest induced by p53, but not p21(Waf1), is associated with a strong reduction in ERK2 activity, phosphorylation, and protein half-life, and with the appearance of caspase activity. Likewise, DNA damage-induced cell cycle arrest correlates with p53-dependent ERK2 downregulation and caspase activation. Furthermore, caspase inhibitors or expression of a caspase-resistant ERK2 mutant interfere with ERK2 cleavage and restore proliferation in the presence of p53 activation, indicating that caspase-mediated ERK2 degradation contributes to p53-induced growth arrest. These findings strongly point to ERK2 as a novel p53 target in growth suppression.

[Surgical treatment of ectopic pregnancy]. / Traitement chirurgical de la grossesse extra-utérine.

Recent advances in therapeutics have considerably modified the management of ectopic pregnancy. However, surgical management is still indicated in some patients. Laparoscopic procedure is the reference surgical approach for the management of ectopic pregnancy. It reduces length of hospital stay, analgesic requirement, hospital cost, the risk of post-surgical adherence and is also associated with lower morbidity and better esthetic appearance. Laparotomy is now only performed in individuals for whom laparoscopy is contra-indicated. Patients can be treated conservatively, tubotomy with removal of the products of conception or radically, salpingectomy. In order to lower the failure rate and the risk of re-occurrence, some operative procedures are required including regular washing of the abdominal cavity, adhesiolysis and extraction in a protective bag. The choice of treatment is generally guided by the patient's past medical history and the wish to bear future children.

[Promontofixation procedure: use of non-absorbable sutures or Tackers?]. / Technique de la promontofixation: suspension au promontoire par fils ou Tackers?

AIM: Description and evaluation of ligamentopexy techniques using strings and spiral staples. MATERIAL AND METHODS: We first describe the ligamentopexy procedure using non-absorbable sutures before comparing this technique to the use of Tacker type staples. We describe the advantages and disadvantages of this procedure in terms of surgical technique, secondary complications and biomechanical strength. RESULTS: For our team, recommendable attitude is to use non-absorbable sutures for the fixation to the prevertebral ligament. The main advantages of the use of staples are the ease and facility for learning the technique. The risk of spondylodiscitis is rare but enhanced by the deeper penetration of the staples into the intervertebral discs. In terms of resistanc, promontofixation using sutures is much stronger compared to staples. CONCLUSION: The use of sutures for promontofixation, in laparoscopy, is preferred to the utilization of staples type Tacker. These staples should be used when there is a risk of needle stitches for the patient.

[Can breast cancer be chemoprevented in 2003?]. / Peut-on prévenir chimiquement le cancer du sein en 2003?

Breast cancer is certainly not preventable at the same degree as lung cancer is by avoiding cigarette smoking or bladder cancer by avoiding exposure to some specific professional carcinogens. Despite the increased availability of screening mammography and the use of adjuvant chemotherapy, breast cancer remains a major cause of morbidity and mortality in women. Chemoprevention could be one of the possible weapons to decrease the morbidity and the mortality due to breast cancer. Experimental, epidemiological and clinical data suggest that one can prevent occurrence of breast cancer. The results of five trials including more than 25,000 patients comparing the effectiveness of tamoxifen with a placebo were published. The results of these trials, at first sight, do not agree. Only in the USA, the results of the five trials have led to licence tamoxifen for chemoprevention of breast cancer. But the Food and Drug Administration stresses the fact that the noxious side effects are too numerous for this molecule to be prescribed with all the patients. It must be reserved for the women presenting an increased risk. The means of identifying the women at increased risk are not yet perfect.

Theories of endometriosis.

Endometriosis is characterised by the presence of abnormally located tissue resembling the endometrium with glands and stroma. Several hypotheses have attempted to explain the development of such tissue. The oldest theory, that of metaplasia, suggests that under diverse influences coelomic tissue could be transformed into endometrium. The most often cited theory, that of implantation, proposes that the physiological phenomenon of endometrial reflux in the fallopian tubes during menstruation may, in certain conditions, overcome local defense mechanisms, implant, and proliferate. The peritoneal fluid in unaffected women possesses the capacity to prevent endometriotic tissue from becoming established. The reasons for the occurrence of endometriosis and its consequences (pain, sterility, adhesions) are probably numerous and involve the endometrium, the immune system (macrophages, natural killer cells), the peritoneum, and fallopian tubes. The failure to clear the peritoneal cavity of fragments of endometrium could cause a state of local inflammation with hyperactivation of macrophages secreting a variety of different compounds. Some of these compounds may bring about metaplasia of the peritoneum or the development of Mullerian residues.

[Ovarian cyst: surgical indications and access]. / Kyste ovarien: indications chirurgicales et voies d'abord.

Laparoscopic treatment of adnexal masses is indicated when all criteria of a benign lesion are present: transvaginal ultrasound demonstrates a mass < 5 cm, with liquid or dermoid content, with less than 3 fine partitions (< 3 mm), a thin wall (< 3 mm), no vegetations, normal Doppler. Laparoscopy is also indicated for "benign" cysts measuring 5 to 10 cm if laparoscopy is feasible. Peroperative exploration is the rule. Since the benign or malignant nature of an ovarian mass cannot be determined macroscopically, precaution must be taken to avoid potential laparoscopic dissemination: use of an extraction pouch, instrument cleaning, cytotoxic agent (chlorexidine or povidone-iodine) for trocar tracts, prevention of gas leakage, 3-plane suture of trocar orifices measuring > 10 mm, short interval between laparoscopic diagnosis of cancer and onset of chemotherapy or complete surgery (1 week). In case of pre- or peroperatively suspected malignancy, cytology examination of the peritoneal fluid and careful peroperative exploration of the abdomen and pelvis with peritoneal biopsy as needed are required. Simple cystectomy or adnexectomy may be performed, depending on the age of the patient, while waiting for the final pathology report. Peroperative intraperitoneal rupture must be avoided, converting to laparotomy if needed. If several suspicious elements are found, median laparotomy is often recommended, particularly in case of suspected cancer with extra-ovarian involvement, or if there is a risk of peroperative rupture. Peroperative pathology of the adnexectomy specimen and peroperative exploration will depend on the operator's experience and the availability of pathology examination in the operating room. First line laparoscopy allows an analysis of the operability and choice of the most appropriate access.

Activation of p53/p21waf1 pathway is associated with senescence during v-Ha-ras transformation of immortal C2C12 myoblasts.

It has recently been shown that tumor cells can retain the ability to undergo senescence, while the capacity of bypassing senescence has been associated with tumor progression. In this report, we showed that v-Ha-ras-mediated transformation of already immortal C2C12 myoblasts can be associated with senesence, in a low amount during in vitro passages and, to a higher extent, affer cellular stress (cell culture alkalinkation), or DNA damage (doxorubicin treatment). The capacity to undergo replicative senescence is associated with a strong increase of wt-p53 transcriptional activity and p21WAF1 up-regulation. These biochemical activities are down-modulated in the cells that evade the massive replicative senescence after stressing stimuli. Altogether, these findings show that active ras can cause senescence during the transformation of already immortal cells in associaton with p53/p21WAF1 pathway activation and support the hypothesis that p53/p21WAF1 functional activity is important in maintaining the integrity of the senescence pathway during cellular transformation.

Exogenous wt-p53 protein is active in transformed cells but not in their non-transformed counterparts: implications for cancer gene therapy without tumor targeting.

BACKGROUND: Expression of exogenous wild-type p53 (wt-p53) protein in tumor cells can suppress the transformed phenotype whereas it does not apparently induce detrimental effects in non-transformed cells. This observation may provide a molecular basis for p53-mediated gene therapy of p53-sensitive cancers without the need for tumor targeting. METHODS: To understand the molecular mechanisms responsible for this different behavior in tumor versus normal cells, biochemical and functional analyses of exogenous wt-p53 protein were performed on non-transformed C2C12 myoblasts and their transformed counterparts, the C2-ras cells. RESULTS: The exogenous wt-p53 protein, which induced persistent growth arrest only in transformed C2-ras cells, was shown to be significantly more stable in transformed than in non-transformed cells. This different stability was due to different p53 proteolytic degradation. Moreover, constitutively, exogenous wt-p53 protein was found to be transcriptionally active only in C2-ras cells but it could also be activated in C2C12 cells by genotoxic damage. CONCLUSIONS: Non-transformed C2C12 cells present regulatory system(s) which control the expression and the activity of exogenously expressed wt-p53 protein probably through degradation and maintenance in a latent form. This regulatory system is lost/inactivated upon transformation.

Increase of BCNU sensitivity by wt-p53 gene therapy in glioblastoma lines depends on the administration schedule.

In this article, we investigated the effect induced by the reintroduction of wild-type p53 (wt-p53) protein on BCNU sensitivity in the ADF glioblastoma line. Using a wt-p53 recombinant adenovirus (Ad-p53), we demonstrated that exogenous wt-p53 expression was able to increase the sensitivity to BCNU in ADF cells. Interestingly, this effect was more evident when Ad-p53 infection was performed after BCNU treatment compared with the opposite sequence. To understand the biological basis of these different behaviors, we analyzed the cell cycle of the differently treated cells. We found that Ad-p53 infection induced a persistent accumulation of cells in the G0/G1 phase while, as expected, BCNU induced a block in the G2-M phase. Ad-p53-->BCNU sequence did not significantly modify the cell cycle profile in respect of Ad-p53 infected cells. In contrast, BCNU-->Ad-p53 sequence provoked G2-M arrest similar to that observed after treatment with BCNU alone, but prevented the later recovery of the cells through the cell cycle, by driving the cells to apoptotic death. These results demonstrate that the administration sequence is important to increase drug sensitivity. To generalize the phenomenon observed on ADF line, the antiproliferative effect of the two different schedules was analyzed on other glioblastoma lines (A172, CRS-A2, U373MG) with different BCNU sensitivity and p53 status. The data obtained confirm that the wt-p53 gene transfer enhances BCNU sensitivity in glioblastoma cells depending on the administration sequence.

Wild-type p53-mediated down-modulation of interleukin 15 and interleukin 15 receptors in human rhabdomyosarcoma cells.

We recently reported that rhabdomyosarcoma cell lines express and secrete interleukin 15 (IL-15), a tightly regulated cytokine with IL-2-like activity. To test whether the p53-impaired function that is frequently found in this tumour type could play a role in the IL-15 production, wild-type p53 gene was transduced in the human rhabdomyosarcoma cell line RD (which harbours a mutated p53 gene), and its effect on proliferation and expression of IL-15 was studied. Arrest of proliferation was induced by wild-type p53; increased proportions of G1-arrested cells and of apoptotic cells were observed. A marked down-modulation of IL-15 expression, at both the mRNA and protein level, was found in p53-transduced cells. Because a direct effect of IL-15 on normal muscle cells has been reported, the presence of IL-15 membrane receptors was studied by cytofluorometric analysis. Rhabdomyosarcoma cells showed IL-15 membrane receptors, which are down-modulated by wild-type p53 transfected gene. In conclusion, wild-type p53 transduction in human rhabdomyosarcoma cells induces the down-modulation of both IL-15 production and IL-15 receptor expression.