A comprehensive preanalytical protocol for fresh solid tumor biospecimens.

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Preanalytical protocol for fresh solid tumor biospecimens.

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. 1-4 These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. 2,5 Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Neutrophil extracellular traps promote MASH fibrosis by metabolic reprogramming of HSC.

BACKGROUND AND AIMS: Metabolic dysfunction-associated steatohepatitis (MASH) fibrosis is a reversible stage of liver disease accompanied by inflammatory cell infiltration. Neutrophils extrude a meshwork of chromatin fibers to establish neutrophil extracellular traps (NETs), which play important roles in inflammatory response regulation. Our previous work demonstrated that NETs promote HCC in MASH. However, it is still unknown if NETs play a role in the molecular mechanisms of liver fibrosis. APPROACH AND RESULTS: Following 12 weeks of Western diet/carbon tetrachloride, MASH fibrosis was identified in C57BL/6 mice with increased NET formation. However, NET depletion using DNase I treatment or mice knocked out for peptidyl arginine deaminase type IV significantly attenuated the development of MASH fibrosis. NETs were demonstrated to induce HSCs activation, proliferation, and migration through augmented mitochondrial and aerobic glycolysis to provide additional bioenergetic and biosynthetic supplies. Metabolomic analysis revealed markedly an altered metabolic profile upon NET stimulation of HSCs that were dependent on arachidonic acid metabolism. Mechanistically, NET stimulation of toll-like receptor 3 induced cyclooxygenase-2 activation and prostaglandin E2 production with subsequent HSC activation and liver fibrosis. Inhibiting cyclooxygenase-2 with celecoxib reduced fibrosis in our MASH model. CONCLUSIONS: Our findings implicate NETs playing a critical role in the development of MASH hepatic fibrosis by inducing metabolic reprogramming of HSCs through the toll-like receptor 3/cyclooxygenase-2/cyclooxygenase-2 pathway. Therefore, NET inhibition may represent an attractive treatment target for MASH liver fibrosis.

Induction of G0/G1 phase cell cycle arrest and apoptosis by thymol through ROS generation and caspase-9/-3 activation in breast and colorectal cancer cell lines.

BACKGROUND: Cancer is a major malignancy and one of the leading causes of death; it calls for a proactive strategy for the cure. Herbs are reservoirs of novel chemical entities and their phytochemical exploration has contributed considerably to the discovery of new anticancer drugs. Thymol, a natural phenolic monoterpenoid, has been implicated with many medicinal properties, including anticancer ones. However, the anti-proliferative and apoptosis-inducing ability of thymol on MDA-MB-231 and HCT-8 cell lines has not been studied yet in detail, and hence this study was conceived. MATERIALS AND METHODS: We studied the cytotoxicity, morphological alterations of the cell, oxidative stress, cell cycle modulation, apoptosis and expression of apoptosis-related proteins that ensued due to thymol treatment in these cancer cells. RESULTS: Thymol inhibited the cell proliferation, altered the morphology of the cells, increased the intracellular ROS level, arrested the cells in G0/G1 phase, induced apoptosis, upregulated pro-apoptotic protein p53 expression, downregulated anti-apoptotic protein Bcl-xL expression, and activated caspase-9 and -3. CONCLUSION: These findings elucidate that thymol induces apoptosis through the intrinsic pathway, in MDA-MB-231 breast and HCT-8 colorectal cancer cells through ROS generation and G0/G1 phase cell cycle arrest. This reiterates the broad-spectrum anti-tumor potential of thymol and provides an insight to study further to be developed into an anticancer drug.

Quinacrine enhances the efficacy of cisplatin by increasing apoptosis and modulating cancer survival proteins in a colorectal cancer cell line.

BACKGROUND: Cisplatin and platinum-based compounds have been used successfully to treat various cancers. However, their use is often restricted due to the acquired resistance by cancer cells. Over-expression of p53 and inhibition of NF-kB sensitize several cancer cells towards cisplatin-induced apoptosis. Quinacrine, a cytotoxic drug with predictable safety revealed to concurrently suppress NF-kB and activate p53, which may be an attractive adjuvant in cisplatin chemotherapy. Therefore, the objective of the present study was to establish the role of quinacrine as an adjuvant in lowering the dose of cisplatin during cancer therapy to circumvent its toxic effects. MATERIALS AND METHODS: The colon cancer (HCT-8) cells were cultured and cell survival assays were performed using standard procedures. Cell cycle arrest and the extent of apoptosis were determined using a muse cell analyzer. Cancer survival proteins were analyzed using western blotting techniques. RESULTS AND CONCLUSION: We demonstrated that concomitant use of quinacrine with cisplatin increased cell apoptosis, suppressed cell proliferation and inhibited colony formation in a colorectal cancer cell line. Moreover, cell cycle arrest in the G0/G1 and G2/M phases and upregulation of p53 expression were observed. There was also downregulation of NF-kB and Bcl-xL protein expressions, both of which are associated with enhanced cell apoptosis and an increase in the sensitivity of cancer cells to cisplatin, overcoming its chemoresistance. Overall, the results of the present study and available literature clearly indicate that the use of quinacrine as an adjuvant with cisplatin may enhance its anti-cancer activity and reduce chemoresistance.

Type 2 Diabetes, Obesity, and Cancer Share Some Common and Critical Pathways.

Diabetes and cancer are among the most frequent and complex diseases. Epidemiological evidence showed that the patients suffering from diabetes are significantly at higher risk for a number of cancer types. There are a number of evidence that support the hypothesis that these diseases are interlinked, and obesity may aggravate the risk(s) of type 2 diabetes and cancer. Multi-level unwanted alterations such as (epi-)genetic alterations, changes at the transcriptional level, and altered signaling pathways (receptor, cytoplasmic, and nuclear level) are the major source which promotes a number of complex diseases and such heterogeneous level of complexities are considered as the major barrier in the development of therapeutic agents. With so many known challenges, it is critical to understand the relationships and the commonly shared causes between type 2 diabetes and cancer, which is difficult to unravel and understand. Furthermore, the real complexity arises from contended corroborations that specific drug(s) (individually or in combination) during the treatment of type 2 diabetes may increase or decrease the cancer risk or affect cancer prognosis. In this review article, we have presented the recent and most updated evidence from the studies where the origin, biological background, the correlation between them have been presented or proved. Furthermore, we have summarized the methodological challenges and tasks that are frequently encountered. We have also outlined the physiological links between type 2 diabetes and cancers. Finally, we have presented and summarized the outline of the hallmarks for both these diseases, diabetes and cancer.

Scorpion Venom Causes Apoptosis by Increasing Reactive Oxygen Species and Cell Cycle Arrest in MDA-MB-231 and HCT-8 Cancer Cell Lines.

OBJECTIVES: The objective of this study was to examine the effect of scorpion venoms on cancer cell progression, apoptosis, and cell cycle arrest. Scorpion venoms are known to possess numerous bioactive compounds that act against cancer progression by inducing apoptosis. In this study, we have taken the venoms from the following 2 species of scorpion- Androctonus crassicauda and Leiurus quinquestriatus-and tested the anticancer properties of the venom against breast and colorectal cancer cell lines. METHODS: Milking of scorpion venom and culturing the breast and colorectal cancer cell lines were done according to the standard procedure. The venom cytotoxicity was assessed by MTT methods, and the cellular and nuclear changes were studied with phase contrast and propidium iodide staining, respectively. The cell cycle arrest and accumulation of reactive oxygen species were analyzed on a Muse cell analyzer. RESULTS: The venoms exerted cytotoxic effects on breast and colorectal cell lines in a dose- and time-dependent manner. Enhanced apoptotic cells, increase in reactive oxygen species, and cell cycle arrest were observed after challenging these cell lines with scorpion venoms. CONCLUSIONS: Scorpion venom induces apoptosis in breast and colorectal cell lines as reflected by the changes in the cell morphology and cell cycle studies. Furthermore, a high percentage of total reactive oxygen species as well as apoptotic cells also contribute to cell death as observed after venom treatments. To the best of authors' knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest by these species of scorpion venoms.

Scorpion Venom Causes Upregulation of p53 and Downregulation of Bcl-xL and BID Protein Expression by Modulating Signaling Proteins Erk1/2 and STAT3, and DNA Damage in Breast and Colorectal Cancer Cell Lines.

Scorpion venoms efficiently block the normal neurotransmitter signaling pathway by prejudicing the ion channel operating mechanism in the body system. Besides its negative effect, venoms also possess some beneficial qualities for humans. They have also been shown to exhibit anticancer properties in various cancer types. This unique property of the venom as an anticancer agent is mainly a result of its role in initiating apoptosis and inhibiting several signaling cascade mechanisms that promote cancer cell proliferation and growth. In this study, we examine the effect of venom on phenotypic changes as well as changes at the molecular levels in colorectal and breast cancer cell lines. A dramatic decrease in cell invasion was observed in both cancer cell lines on venom treatment. Additionally, there was decrease in IL-6, RhoC, Erk1/2, and STAT3 in venom-treated cell lines, providing strong evidence of its anticancer properties. Furthermore, decrease in the expression of antiapoptotic proteins and also upregulation of proapoptotic ones by these lines were observed on venom treatment. Moreover, a vivid picture of DNA damage was also detected on venom treatment. In conclusion, scorpion venom possesses significant potential as an anticancer agent against colorectal and breast cancer cell lines.

In vitro determination of the efficacy of scorpion venoms as anti-cancer agents against colorectal cancer cells: a nano-liposomal delivery approach.

The use of liposomes in biological and medicinal sciences is a relatively new approach. The liposomal strategy greatly depends on the technological advancement in the formation of vesicles of various sizes and properties. In the current study, we encapsulated the venoms obtained from medically important scorpions such as Androctonus bicolor (AB), Androctonus crassicauda (AC), and Leiurus quinquestriatus (LQ). To begin with, our first and foremost aim was to prepare biocompatible and biodegradable nanovesicles. Additionally, we intended to enhance the anti-cancer potential of these encapsulated venoms. The liposomal venoms were prepared by rehydration and dehydration methods. Morphology, particle size, and size distribution of the liposomes were examined by scanning electron microscope (SEM), transmission electron microscope (TEM), and Zetasizer. We found that the prepared liposomes had a smooth surface and a spherical/ovoid shape and existed mainly as single unilamellar vesicles (SUVs). Furthermore, the liposomal formulation of all three venoms exhibited excellent stability and good encapsulation efficiency (EE). Additionally, the anti-cancer potential of the encapsulated venoms was also evaluated on a colorectal cancer cell line (HCT-8). The venom-loaded liposomes showed elevated anti-cancer properties such as low rate of cell survival, higher reactive oxygen species (ROS) generation, and enhancement in the number of apoptotic cells. In addition to this, cell cycle analysis revealed G0/G1 enrichment upon venom treatment. The effect of treatment was more pronounced when venom-liposome was used as compared to free venom on the HCT-8 cell line. Furthermore, we did not observe any interference of liposomal lipids used in these preparations on the progression of cancer cells. Considering these findings, we can conclude that the encapsulated scorpion venoms exhibit better efficacy and act more vigorously as an anti-cancer agent on the colorectal cancer cell line when compared with their free counterpart.

Snake venom causes apoptosis by increasing the reactive oxygen species in colorectal and breast cancer cell lines.

Snake venom possesses various kinds of proteins and neurotoxic polypeptides, which can negatively interfere with the neurotransmitter signaling cascade. This phenomenon occurs mainly due to the blocking of ion channels in the body system. Envenomation prevents or severely interrupts nerve impulses from being transmitted, inhibition of adenosine triphosphate synthesis, and proper functioning of the cardiac muscles. However, some beneficial properties of venoms have also been reported. The aim of this study was to examine the snake venom as an anticancer agent due to its inhibitory effects on cancer progression such as cell motility, cell invasion, and colony formation. In this study, the effect of venoms on phenotypic changes and the change on molecular level in colorectal and breast cancer cell lines were examined. A reduction of 60%-90% in cell motility, colony formation, and cell invasion was observed when these cell lines were treated with different concentrations of snake venom. In addition, the increase in oxidative stress that results in an increase in the number of apoptotic cancer cells was significantly higher in the venom-treated cell lines. Further analysis showed that there was a decrease in the expression of pro-inflammatory cytokines and signaling proteins, strongly suggesting a promising role for snake venom against breast and colorectal cancer cell progression. In conclusion, the snake venoms used in this study showed significant anticancer properties against colorectal and breast cancer cell lines.

Suberoylanilide hydroxamic acid inhibits growth of head and neck cancer cell lines by reactivation of tumor suppressor microRNAs.

BACKGROUND: microRNAs negatively regulate gene expression at the post-transcriptional level. Mounting evidence shows that miR expression is deregulated in human cancers including head and neck squamous cell carcinoma (HNSCC). Epigenetically silenced tumor suppressor miRs may be re-expressed upon treatment with histone deacetylases inhibitors. Suberoylanilide Hydroxamic Acid (SAHA) is a histone deacetylase inhibitor that is currently being investigated in clinical trials for HNSCC. We hypothesized that SAHA will re-express a set of tumor suppressor miRs and enhance the efficacy of cisplatin and radiation in HNSCC. RESULTS: In this study, miR expression profile was utilized to identify the tumor suppressor miRs that are re-expressed following SAHA treatment in HNSCC. Our data demonstrated that two tumor suppressor miRs, miR-107 and miR-138, were significantly up-regulated in CAL27 and SCC25 cell lines, following SAHA treatment. In addition to this, treatment with SAHA in a dose dependent manner significantly inhibited the cell proliferation, cell migration, and anchorage dependent clonogenic survival in CAL27 and SCC25 cell lines, respectively. Further, the expression of several oncogenes, PKCε, HIF1ß, CDK6, and RhoC were down regulated in response to SAHA treatment. Additionally, we demonstrated that the combination treatment with SAHA and a chemotherapeutic drug cisplatin caused a significant reduction of cell growth compared to the single agent treatment. CONCLUSION: Our data indicate that SAHA treatment results in reactivation of the silenced tumor suppressor miRs. Furthermore, this study emphasizes the usefulness of this drug as a novel combination therapy for HNSCC patients.

In vitro analysis of the anticancer properties of scorpion venom in colorectal and breast cancer cell lines.

Scorpion venom contains various types of proteins and peptides that are able to act as inhibitors of neurotransmitter molecules. This is achieved primarily via the inhibition of ion channels. In addition, scorpion venom has been demonstrated to exhibit anticancer properties in prostate and breast cancer, as well as leukemia. The anticancer properties of scorpion venom are due to its inhibitory effect on matrix metalloproteinase (MMP) activity, which leads to reduced motility and invasion in tumor cells. The inhibitory effects of venom on MMPs additionally lead to a reduction in the metastatic potential of malignant tumors. In the present study, the effect of venom obtained from a local serpentarium facility was examined in colorectal and breast cancer cell lines. Cell motility and clonogenic survival assays revealed a significant decrease (60-90%) in cell motility and colony formation, two significant hallmarks of cancer survival, following treatment with various concentrations of venom. These results were in agreement with previous studies demonstrating the anticancer activity of scorpion venom. In conclusion, the venom utilized at the Research Center of Prince Sultan Military Medical City Hospital (Riyadh, Saudi Arabia) possesses significant anticancer potential against colorectal and breast cancer cell lines.

Moringa oleifera as an Anti-Cancer Agent against Breast and Colorectal Cancer Cell Lines.

In this study we investigated the anti-cancer effect of Moringa oleifera leaves, bark and seed extracts. When tested against MDA-MB-231 and HCT-8 cancer cell lines, the extracts of leaves and bark showed remarkable anti-cancer properties while surprisingly, seed extracts exhibited hardly any such properties. Cell survival was significantly low in both cells lines when treated with leaves and bark extracts. Furthermore, a striking reduction (about 70-90%) in colony formation as well as cell motility was observed upon treatment with leaves and bark. Additionally, apoptosis assay performed on these treated breast and colorectal cancer lines showed a remarkable increase in the number of apoptotic cells; with a 7 fold increase in MD-MB-231 to an increase of several fold in colorectal cancer cell lines. However, no significant apoptotic cells were detected upon seeds extract treatment. Moreover, the cell cycle distribution showed a G2/M enrichment (about 2-3 fold) indicating that these extracts effectively arrest the cell progression at the G2/M phase. The GC-MS analyses of these extracts revealed numerous known anti-cancer compounds, namely eugenol, isopropyl isothiocynate, D-allose, and hexadeconoic acid ethyl ester, all of which possess long chain hydrocarbons, sugar moiety and an aromatic ring. This suggests that the anti-cancer properties of Moringa oleifera could be attributed to the bioactive compounds present in the extracts from this plant. This is a novel study because no report has yet been cited on the effectiveness of Moringa extracts obtained in the locally grown environment as an anti-cancer agent against breast and colorectal cancers. Our study is the first of its kind to evaluate the anti-malignant properties of Moringa not only in leaves but also in bark. These findings suggest that both the leaf and bark extracts of Moringa collected from the Saudi Arabian region possess anti-cancer activity that can be used to develop new drugs for treatment of breast and colorectal cancers.

Down regulation of RhoC by microRNA-138 results in de-activation of FAK, Src and Erk1/2 signaling pathway in head and neck squamous cell carcinoma.

OBJECTIVE: RhoC a pro-metastatic oncogene is constitutively active in many head and neck squamous cell carcinomas. MicroRNA-138 which possesses a documented tumor suppressor function can bind to the 3'UTR of RhoC mRNA and inhibit its activity. We hypothesize that miR-138 can inhibit the function of RhoC and consequently the activation of downstream target molecules involve in the signaling cascade. For this reason we investigated the role of miR-138 in HNSCC. METHODS: In vitro studies were carried out to evaluate the role of miR-138 in HNSCC cell lines and in primary tumors obtained from HNSCC patients. Real time RT-PCR, Western blot, cell motility, invasion and colony formation assays were performed according to standard procedures. RESULTS: Data obtained by G-LISA and real time PCR shows an inverse correlation between RhoC expression and miR-138 in HNSCC cell lines. Additionally, we obtained a similar pattern of RhoC and miR-138 expression in primary tumors from HNSCC patients. Over expression of miR-138 in HNSCC lines showed down regulation of RhoC, as well as a decrease in cell motility, invasion colony and stress fiber formation. Furthermore, a significant down regulation was observed for FAK, Src and Erk(1/2) upon miR-138 overexpression. CONCLUSION: These findings strongly suggest that the inhibition of RhoC can be achieved by over expressing miR-138, which further attenuates the downstream signaling cascade leading to cancer progression and survival. Moreover, this study for the first time shows that down regulation of FAK, Src and Erk(1/2) by miR-138 overexpression is due to inhibition of RhoC in HNSCC.

RhoC regulates cancer stem cells in head and neck squamous cell carcinoma by overexpressing IL-6 and phosphorylation of STAT3.

In this study we investigated the correlation between RhoC expression and cancer stem cells (CSCs) formation in head and neck squamous cell carcinoma (HNSCC). The inhibition of RhoC function was achieved using shRNA. The expression of stem cell surface markers, ALDH and CD44 were significantly low in two RhoC depleted HNSCC cell carcinoma cell lines. Furthermore, a striking reduction in tumorsphere formation was achieved in RhoC knockdown lines. The mRNA expression of RhoC in RhoC knockdown adherent and tumorspheres are dramatically down regulated as compared with the scrambled control. The mRNA expression of stem cell transcription factors; nanog, oct3/4 (Pouf1), and sox2 were significantly depleted in RhoC knockdown clones. Further, the phosphorylation of STAT3(ser727), and STAT3(tyr705) were significantly down regulated in RhoC knockdown clones. The overexpression of STAT3 in RhoC knockdown did not show any change in expression patterns of either-STAT3(tyr705) or stem cell transcription factors, signifying the role of RhoC in STAT3 activation and thus the expression of nanog, oct3/4 and sox2 in HNSCC. The expression of Inter leukin-6 (IL-6) in RhoC knockdown HNSCC cell lines was dramatically low as compared to the scrambled control. Further, we have shown a rescue in STAT3 phosphorylation by IL-6 stimulation in RhoC knockdown lines. This study is the first of its kind to establish the involvement of RhoC in STAT3 phosphorylation and hence in promoting the activation of core cancer stem cells (CSCs) transcription factors. These findings suggest that RhoC may be a novel target for HNSCC therapy.

Atorvastatin inhibits RhoC function and limits head and neck cancer metastasis.

OBJECTIVE: RhoC oncogene is a well characterized marker of metastasis in a majority of invasive cancers, including HNSCC. Elevated RhoC expression has been found to be associated with distant metastasis. Statins are a class of drugs that are used to reduce cholesterol levels by inhibiting HMG-CoA reductase activity which in turns prevents mevalonate synthesis, which is a precursor for synthesis of cholesterol and prenylation. Interestingly, the proper function of Rho proteins depends on prenylation. Significantly, it has been reported that metastasis in human melanoma can be reduced by atorvastatin which inhibits RhoC activity by preventing its geranylgeranylation. Given that RhoC is a key oncogene involved in metastasis, we hypothesized Atorvastatin can reduce head and neck metastasis by inhibiting RhoC activity. METHODS: In vitro and in vivo studies were carried out to evaluate the ability of Atorvastatin to inhibit RhoC function and HNSCC metastasis. Cell motility, proliferation, cell invasion, and colony formation assays were performed according to the standard protocols. RESULTS: Atorvastatin treatment significantly reduced the active form of RhoC in vitro and diminished cell motility, invasion, proliferation and colony formation. Importantly, we observed a significant decrease in p-ERK1/2 and p-STAT3 in Atorvastatin treated cell lines. In vivo experiments revealed inhibition of angiogenesis and lung metastases with Atorvastatin therapy. CONCLUSIONS: This study is the first of its kind to establish a potential role of Atorvastatin in head and neck cancer therapy. These findings suggest that Atorvastatin can be a potential low risk adjuvant therapy to minimize metastases in aggressive forms of HNSCC.

Health behaviors predict higher interleukin-6 levels among patients newly diagnosed with head and neck squamous cell carcinoma.

BACKGROUND: Health behaviors have been shown to be associated with recurrence risk and survival rates in patients with cancer and are also associated with interleukin-6 (IL-6) levels, but few epidemiologic studies have investigated the relationship of health behaviors and IL-6 among cancer populations. The purpose of the study is to look at the relationship between five health behaviors, viz.: smoking, alcohol problems, body mass index (BMI; a marker of nutritional status), physical activity, and sleep and pretreatment IL-6 levels in persons with head and neck cancer. METHODS: Patients (N = 409) were recruited in otolaryngology clinic waiting rooms and invited to complete written surveys. A medical record audit was also conducted. Descriptive statistics and multivariate analyses were conducted to determine which health behaviors were associated with higher IL-6 levels controlling for demographic and clinical variables among patients with newly diagnosed head and neck cancer. RESULTS: While smoking, alcohol problems, BMI, physical activity, and sleep were associated with IL-6 levels in bivariate analysis, only smoking (current and former) and decreased sleep were independent predictors of higher IL-6 levels in multivariate regression analysis. Covariates associated with higher IL-6 levels were age and higher tumor stage, whereas comorbidities were marginally significant. CONCLUSION: Health behaviors, particularly smoking and sleep disturbances, are associated with higher IL-6 levels among patients with head and neck cancer. IMPACT: Treating health behavior problems, especially smoking and sleep disturbances, may be beneficial to decreasing IL-6 levels, which could have a beneficial effect on overall cancer treatment outcomes.

Tetrathiomolybdate inhibits head and neck cancer metastasis by decreasing tumor cell motility, invasiveness and by promoting tumor cell anoikis.

BACKGROUND: The metastatic spread of solid tumors is directly or indirectly responsible for most cancer-related deaths. Tumor metastasis is very complex and this process requires a tumor cell to acquire enhanced motility, invasiveness and anoikis resistance to successfully establish a tumor at a distal site. Metastatic potential of tumor cells is directly correlated with the expression levels of several angiogenic cytokines. Copper is a mandatory cofactor for the function of many of these angiogenic mediators as well as other proteins that play an important role in tumor cell motility and invasiveness. We have previously shown that tetrathiomolybdate (TM) is a potent chelator of copper and it mediates its anti-tumor effects by suppressing tumor angiogenesis. However, very little is known about the effect of TM on tumor cell function and tumor metastasis. In this study, we explored the mechanisms underlying TM-mediated inhibition of tumor metastasis. RESULTS: We used two in vivo models to examine the effects of TM on tumor metastasis. Animals treated with TM showed a significant decrease in lung metastasis in both in vivo models as compared to the control group. In addition, tumor cells from the lungs of TM treated animals developed significantly smaller colonies and these colonies had significantly fewer tumor cells. TM treatment significantly decreased tumor cell motility and invasiveness by inhibiting lysyl oxidase (LOX) activity, FAK activation and MMP2 levels. Furthermore, TM treatment significantly enhanced tumor cell anoikis by activating p38 MAPK cell death pathway and by downregulating XIAP survival protein expression. CONCLUSIONS: Taken together, these results suggest that TM is a potent suppressor of head and neck tumor metastasis by modulating key regulators of tumor cell motility, invasiveness and anoikis resistance.

RhoC expression and head and neck cancer metastasis.

RhoC protein, a known marker of metastases in aggressive breast cancers and melanoma, has also been found to be overexpressed in certain head and neck cancers, thus we investigated the correlation between RhoC expression and the metastatic behavior of head and neck squamous cell carcinoma. Selective inhibition of RhoC expression was achieved using lentiviral small hairpin RNA (shRNA) transduced and tracked with green fluorescent protein to achieve 70% to 80% RhoC inhibition. Fluorescence microscopy of the RhoC knockdown stable clones showed strong green fluorescence in the majority of cells, signifying a high efficiency of transduction. Importantly, quantitative real-time PCR showed no significant decrease in the mRNA expression levels of other members of the Ras superfamily. Cell motility and invasion were markedly diminished in RhoC-depleted cell lines as compared with control transduced lines. H&E staining of lung tissue obtained from severe combined immunodeficiency mice, which had been implanted with RhoC knockdown cells, showed a marked decrease in lung metastasis and inflammation of the blood vessels. The cultured lung tissue showed a significant decrease in cell growth in mice implanted with RhoC-depleted cell lines as compared with shRNA-scrambled sequence control lines. Microscopic studies of CD31 expression revealed substantial quantitative and qualitative differences in the primary tumor microvessel density as compared with parental and shRNA-scrambled controls. This study is the first of its kind to establish the involvement of RhoC specifically in head and neck metastasis. These findings suggest that RhoC warrants further investigation to delineate its robustness as a novel potentially therapeutic target.

Interleukin-6 predicts recurrence and survival among head and neck cancer patients.

BACKGROUND: Increased pretreatment serum interleukin (IL)-6 levels among patients with head and neck squamous cell carcinoma (HNSCC) have been shown to correlate with poor prognosis, but sample sizes in prior studies have been small and thus unable to control for other known prognostic variables. METHODS: A longitudinal, prospective cohort study determined the correlation between pretreatment serum IL-6 levels, and tumor recurrence and all-cause survival in a large population (N = 444) of previously untreated HNSCC patients. Control variables included age, sex, smoking, cancer site and stage, and comorbidities. Kaplan-Meier plots and univariate and multivariate Cox proportional hazards models were used to study the association between IL-6 levels, control variables, and time to recurrence and survival. RESULTS: The median serum IL-6 level was 13 pg/mL (range, 0-453). The 2-year recurrence rate was 35.2% (standard error, 2.67%). The 2-year death rate was 26.5% (standard error, 2.26%). Multivariate analyses showed that serum IL-6 levels independently predicted recurrence at significant levels [hazard ratio (HR) = 1.32; 95% confidence interval (CI), 1.11 to 1.58; P = .002] as did cancer site (oral/sinus). Serum IL-6 level was also a significant independent predictor of poor survival (HR = 1.22; 95% CI, 1.02 to 1.46; P = .03), as were older age, smoking, cancer site (oral/sinus), higher cancer stage, and comorbidities. CONCLUSIONS: Pretreatment serum IL-6 could be a valuable biomarker for predicting recurrence and overall survival among HNSCC patients. Using IL-6 as a biomarker for recurrence and survival may allow for earlier identification and treatment of disease relapse.