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.

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.

MDR-1 polymorphisms (G2677T and C3435T) in B-chronic lymphocytic leukemia: an impact on susceptibility and prognosis.

Patients with B-chronic lymphocytic leukemia present diverse clinical features, genetic abnormalities, variable response to treatment, and heterogeneous prognosis. Novel biological markers such as IgVH mutation, CD38, and ZAP-70 expression have shown to offer important prognostic information. An altered expression of the multidrug resistance 1 may represent an additional prognostic marker. Aim of our study was to evaluate two MDR-1 gene polymorphisms: G2677T polymorphism in exon 21 and C3435T polymorphism in exon 26, to evidence if polymorphisms influence the risk of development of B-CLL and whether genomic polymorphisms provide prognostic information on the clinical progression of the disease. A total of 125 patients with B-CLL and 125 healthy subjects were enrolled in this study. The mutant homozygous 2677 TT genotype was found to be associated with the occurrence of B-CLL and higher T allele frequency in patients with B-CLL when compared with controls was observed (P=0.009). When comparing the prognostic patients' characteristics, patients with 2677 GT genotype were statistically linked to the unmutated IgVH genes (r=0.209, P=0.01). Moreover, the same genotype was correlated with lymphocyte number (r=0.269, P=0.02). Finally for the 2677GT polymorphism, the heterozygous status was associated with higher hemoglobin levels (r=0.247, P=0.005). As far the C3435T MDR1 polymorphism, we were not able to identify any significant correlation with IgVH gene status or other variables. In conclusion, MDR1 gene polymorphism could be a factor predisposing to LLC. Moreover, our findings support the possibility of considering these genomic polymorphisms as prognostic markers in patients with B-CLL.

Neuropathic pain in temporomandibular joint disorders: case-control analysis by MR imaging.

BACKGROUND AND PURPOSE: Temporomandibular joint disorders (TMJ-D) may be associated with the onset of neuropathic pain. The purpose of this study was to prospectively assess if, at the open-mouth position, the distance between the temporomandibular joint (TMJ) disk and the mandibular nerve is shorter in patients with TMJ-D and neuropathic pain vs patients with TMJ-D without neuropathic pain or in healthy people. MATERIALS AND METHODS: After ethical committee approval, we evaluated by MR imaging 16 TMJs with TMJ-D and neuropathic pain, 16 TMJs with TMJ-D without neuropathic pain, and 16 TMJs of healthy volunteers. All of the subjects were informed about the study procedure. We evaluated the distance between the TMJ disk and the mandibular nerve at the oval foramen level. Furthermore, the presence within the TMJs of internal derangement, osteoarthrosis, joint effusion, and bone marrow edema was evaluated. RESULTS: At the maximal open-mouth position, the distance between the TMJ disk and the mandibular nerve is shorter in patients with TMJ-D and neuropathic pain than in patients with TMJ-D without neuropathic pain or in healthy volunteers (P < .05). The imaging findings of TMJ internal derangement, effusion, osteoarthrosis, and bone marrow edema were present both in patients with TMJ-D without neuropathic pain and in patients with TMJ-D and neuropathic pain. CONCLUSIONS: We suggest that a closer proximity between the TMJ disk and the mandibular nerve could be one of the causes of the onset of neuropathic pain in patients with TMJ-D and neuropathic pain.

Morphometric evaluation of the temporomandibular joint and the masticatory spaces: the role of high-definition MRI.

AIM: The aim of this work was to describe the anatomy of the temporomandibular joint (TMJ), the masticator spaces and their possible variance by means of magnetic resonance imaging MRI. METHODS: We evaluated one TMJ in each of 28 volunteers (14 males and 14 females) without temporomandibular disorders. The TMJ with mouth closed was evaluated on axial, coronal and sagittal planes obtained with a 1.5 Tesla magnetic resonance scanner. MRI scans of the anatomic patterns of the temporomandibular region were analyzed and compared with the classical anatomy notions reported in the literature. Morphometric evaluation of the temporomandibular region and the medial and lateral pterygoid muscles was carried out. RESULTS: MRI anatomy of the TMJ, the masticator spaces and their possible variance were accurately described. In addition, morphometric evaluation of the TMJ and the masticator spaces was performed. Statistical analysis of the measurements showed that the length of the structures related to the glenoid fossa were usually longer in the female than in the males, whereas the condyle and masticator muscles were usually greater in the males than in the females. T-tests showed no significant differences (P > 0.05) between the measurements of the right and left TMJ. CONCLUSIONS: In preoperative maxillofacial surgery assessment, MRI is a useful tool to reduce operating time, avert surgical complications and improve patient outcome.

[Computed tomography evaluation of urothelial carcinomas of the upper urinary tract]. / Valutazione con Tomografia Computerizzata dei carcinomi uroteliali delle vie escretrici urinarie superiori.

Computed Tomography (CT) is an indispensable noninvasive method for staging transitional cell carcinomas of renal pelvis and ureter. Twenty-seven patients with upper urinary tract tumors were examined and the CT results correlated with histopathologic findings. These tumors exhibit three different development patterns: in our series we identified 17 intraluminal sessile lesions, 6 focal or concentric wall thickenings and finally 7 infiltrating masses; soft-tissue density lesions exhibited in all cases mild contrast enhancement (mean density increase: 38 HU). Even though the attenuation values of tumors do not allow the accurate demonstration of the depth of renal pelvis and ureteral wall invasion, CT can differentiate the lesions which are still confined within the external wall layers (CT1, 17 cases) from those invading the peripelvic-ureteral fat (CT2, 9 cases) or spreading to other organs as well as distant metastases (CT3, 4 cases). Delayed scans can also demonstrate intraluminal lesion extent and sometimes changes of patients' position allow better differentiation of wall outlines from peripheral fat. The overall staging accuracy of CT was 76.66% and its sensitivity was 93.33%. The assessment of lymph node metastases was the major cause of error in the CT evaluation of these tumors.

[Magnetic resonance in the study of the facial nerve: methodological approach and normal anatomy]. / La risonanza magnetica nello studio del nervo facciale: approccio metodologico ed anatomia normale.

The authors report their experience in the study of facial nerve anatomy by means of MR Imaging. The seventh pair of cranial nerves was studied in 6 healthy and informed volunteers with a super-conductive MR unit at 0.5 T using surface and head coils. Slices were 3 mm thick and were acquired on the axial and sagittal planes, when the petrous and the mastoid bones were studied. The parotid gland was studied with 5-mm slices acquired on the axial, angled axial, and sagittal planes. In all cases T1-weighted images (TR 450, TE 30) were performed. If the above research protocol, which is relatively simple and direct, is applied, the whole course of the facial nerve up to its main distal branches can be almost completely demonstrated.

[Magnetic resonance in the evaluation of parametrial involvement in carcinoma of the cervix uteri]. / La risonanza magnetica nella valutazione del coinvolgimento parametriale nel carcinoma della cervice uterina.

Cervical carcinoma is one of the most frequent gynecologic malignancies. Its prognosis depends on both tumor volume at diagnosis and its stage. Staging accuracy is important not only for prognosis but also for optimal treatment planning. According to FIGO criteria, carcinomas without parametrial involvement (stage I and limited stage IIA disease) can be surgically treated. For more advanced stages, treatment, in most cases, consists of radiation therapy or chemotherapy alone. The authors evaluated MR accuracy in the diagnosis of parametrial involvement; to this purpose, 32 patients with histologically proven lesions were referred for MR imaging, which was performed with a 0.5 T superconductive magnet. Transverse and sagittal SE images were obtained with T2 weighting (TR 1800 ms, TE 30-100 ms); transverse and sometimes sagittal images were obtained with T1 weighting (TR 450/300 ms, TE 20/30). T1-weighted images distinguished neoplasm from cervical stroma or dense parametrial connective tissue in 40% of cases only. T2-weighted images, instead, demonstrated the difference in all cases, showing tumor as a hyperintense area in 90% of patients. Neoplastic involvement of pericervical connective tissue was diagnosed, with those sequences, on the basis of focal disruptions of the outer hypointense fibrous cervical stroma; findings were correlated with those from a previous clinical staging and in 26/32 patients with pathologic findings. MR accuracy in demonstrating parametrial involvement was 88%, sensitivity was 77% and specificity was 94%. Clinical staging accuracy in the evaluation of this parameter was 66%. In 6 cases with no surgical findings, MR confirmed extensive parametrial and vesical or rectal neoplastic involvement, as diagnosed at clinics. MR imaging, thanks to its multiplanar and multiparametric imaging capabilities is a very reliable technique in the preoperative staging of cervical carcinoma. Moreover, since clinical staging can sometimes underestimate pericervical connective spread, the higher accuracy of MR imaging can help avoid useless interventional procedures.