Curcumin inhibits human cancer cell growth and migration through downregulation of SVCT2.

Curcumin is a natural polyphenol that is extracted from the rhizomes of the turmeric plant (Curcuma longa), a member of the ginger family. It has been used for centuries in traditional Indian and Chinese medicine for its medicinal properties, including anti-inflammatory, antioxidant and antitumor effects. SVCT2 (Solute Carrier Family 23 Member 2, also known as SLC23A2) is a protein that plays a role in the transport of Vitamin C (Ascorbic Acid) into cells. SVCT2 plays an important role in tumor progression and metastasis, however, the molecular mechanisms of curcumin on SVCT2 have not been studied to date. Curcumin treatment inhibited proliferation and migration of cancer cells in a dose dependent manner. We found that curcumin reduced the expression of SVCT2 in cancer cells with a wild type p53, but not in those with a mutant type of p53. SVCT2 downregulation also reduced the MMP2 activity. Taken together, our results indicate that curcumin inhibited human cancer cell growth and migration by regulating SVCT2 through a downregulating p53. These findings provide new insights into the molecular mechanisms of curcumin's anticancer effects and potential therapeutic strategies for the treatment of metastatic migration.

Proteomic profiling of lymphedema development in mouse model.

The lymphatic vascular system plays an important role in tissue fluid homeostasis. Lymphedema is a chronic, progressive, and incurable condition that leads to lymphatic fluid retention; it may be primary (heritable) or secondary (acquired) in nature. Although there is a growing understanding of lymphedema, methods for the prevention and treatment of lymphedema are still limited. In this study, we investigated differential protein expressions in sham-operated and lymphedema-operated mice for 3 days, using two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis. Male improved methodology for culturing noninbred (ICR) mice developed lymphedema in the right hindlimb. Twenty functional proteins were found to be differentially expressed between lymphedema induced-right leg tissue and normal left leg tissue. Out of these proteins, the protein levels of apolipoprotein A-1 preprotein, alpha-actinin-3, mCG21744, parkinson disease, serum amyloid P-component precursor, annexin A8, mKIAA0098 protein, and fibrinogen beta chain precursor were differentially upregulated in the lymphedema mice compared with the sham-operated group. Western blotting analysis was used to validate the proteomics results. Our results showing differential up-regulation of serum amyloid P-component precursor, parkinson disease, and apolipoprotein A-1 preprotein in lymphedema model over sham-operated model suggest important insights into pathophysiological target for lymphedema. Copyright © 2016 John Wiley & Sons, Ltd.

Oxidative stress level is not associated with survival in terminally ill cancer patients: a preliminary study.

BACKGROUND: While cancer patients have higher oxidative stress (OS) and lower antioxidant activity, evidence for the association of these parameters with survival in patients with terminally ill cancer is lacking. METHODS: We followed 65 terminal cancer patients prospectively. We assessed their performance status, some symptoms, and serum levels of vitamin C and OS level. The Gehan's generalized Wilcoxon test was used to examine the association between survival times and variables. RESULTS: Subjects' performance status was very poor and they had a high level of OS and a low level of vitamin C. No significant association of these two parameters with survival time was noted (p-value, 0.637 for high OS and 0.240 for low vitamin C). Poor performance status was independently related to high OS status after adjusting for potential confounders (adjusted OR, 4.45; p-value, 0.031). CONCLUSIONS: In this study, OS was not associated with survival of terminally ill cancer patients and its prognostic role requires further study.

Phloretin Inhibits the Proliferation of Breast Cancer Cells Through the Down-regulation of Estrogen Receptor α.

BACKGROUND/AIM: Phloretin is a natural flavonoid compound found in some plants, such as apples and pears, as well as in the bark of apple trees. Phloretin has been shown to have inhibitory effects on glucose transporters in cells and can potentially inhibit the growth of cancer cells. However, the mechanism by which phloretin regulates the expression of estrogen receptor alpha (ERα), a key transcription factor in breast cancer, is still unclear. This study investigated how phloretin affects the growth of ERα positive human breast cancer cells. MATERIALS AND METHODS: The growth of breast cancer cell lines, including MCF7 and T47D, was examined using cell proliferation and colony formation assays. Western blotting and semi-quantitative RT-PCR were used to examine protein and mRNA levels, respectively. Localization of cellular proteins was analyzed using subcellular fractionation. Transient transfection and reported gene assays were used to elucidate the impact of phloretin on cell proliferation and ERα transactivation. RESULTS: Phloretin decreased ERα expression at the mRNA and protein levels in MCF7 and T47D cells. It also inhibited the binding of ERα to the estrogen response element present in the promoter of target genes. Moreover, treatment with phloretin inhibited the expression of cyclin D1 and breast cancer marker gene pS2, which are known ERα target genes. Consequently, it inhibited the growth of ERα-positive human breast cancer cells. Furthermore, inhibition of breast cancer growth by phloretin was found to be mediated through both the ERα and ERK1/ERK2 pathways. CONCLUSION: Phloretin, a dihydrochalcone extracted from natural sources, exhibits the ability to regulate ERα function and suppress breast cancer cell proliferation.

Butein derivatives prevent obesity and improve insulin resistance through the induction of energy expenditure in high-fat diet-fed obese mice.

Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3 s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3 s at 5 mg/kg prevented weight gain by 13.6 % in high-fat diet-fed mice without any toxicological observation. In addition, compound 3 s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T1/2), and blood exposure ratio of i.p to i.v were significantly improved in compound 3 s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3 s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3 s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation.

Interleukin-6 but not tumour necrosis factor-alpha predicts survival in patients with advanced cancer.

PURPOSE: The purpose of this study was to evaluate the prognostic role of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in the survival of patients with advanced cancer. METHODS: In this prospective cohort study between three hospice and palliative care centres in South Korea, we followed 98 advanced cancer patients until death or the end of the study. Approximately 60 % of the patients had poor functional status (Eastern Cooperative Oncology Group score ≥3). We investigated the symptoms of cancer cachexia anorexia syndrome, possible cytokine-related confounders such as infection and medication records. Influence from clinical variables was adjusted using the Cox proportional hazard model. RESULTS: The median survival time was 27 days. On multivariate analysis, elevated IL-6 (hazard ratio, 2.139; p = 0.003) was found to be an independent significant prognostic factor. TNF-α was not a significant factor. Poor performance status and male gender were also independently related to shortened survival. CONCLUSIONS: IL-6 level can be a useful indicator of survival time of patients with advanced cancer at the very end of life. In contrast, the prognostic role of TNF-α requires further study.

Intravenous vitamin C administration reduces fatigue in office workers: a double-blind randomized controlled trial.

BACKGROUND: Studies of the efficacy of vitamin C treatment for fatigue have yielded inconsistent results. One of the reasons for this inconsistency could be the difference in delivery routes. Therefore, we planned a clinical trial with intravenous vitamin C administration. METHODS: We evaluated the effect of intravenous vitamin C on fatigue in office workers. A group of 141 healthy volunteers, aged 20 to 49 years participated in this randomized, double-blind, controlled clinical trial. The trial group received 10 grams of vitamin C with normal saline intravenously, while the placebo group received normal saline only. Since vitamin C is a well-known antioxidant, oxidative stress was measured. Fatigue score, oxidative stress, and plasma vitamin C levels were measured before intervention, and again two hours and one day after intervention. Adverse events were monitored. RESULTS: The fatigue scores measured at two hours after intervention and one day after intervention were significantly different between the two groups (p = 0.004); fatigue scores decreased in the vitamin C group after two hours and remained lower for one day. Trial also led to higher plasma vitamin C levels and lower oxidative stress compared to the placebo group (p < 0.001, p < 0.001, respectively). When data analysis was refined by dividing each group into high-baseline and low-baseline subgroups, it was observed that fatigue was reduced in the lower baseline vitamin C level group after two hours and after one day (p = 0.004). The same did not hold for the higher baseline group (p = 0.206). CONCLUSION: Thus, intravenous vitamin C reduced fatigue at two hours, and the effect persisted for one day. There were no significant differences in adverse events between two groups. High dose intravenous vitamin C proved to be safe and effective against fatigue in this study. TRIAL REGISTRATION: The clinical trial registration of this trial is http://ClinicalTrials.govNCT00633581.

The relationship between interleukin-6, tumor necrosis factor-α, and fatigue in terminally ill cancer patients.

Although many cancer patients receiving palliative care experience distressing levels of fatigue, no well-designed studies have investigated contributing factors in Korean patients. We conducted a cross-sectional study using the Brief Fatigue Inventory-K (BFI-K) to measure fatigue while assessing a variety of possible correlates. Ninety patients with incurable cancer in the terminal stage (median survival: 27 days) participated in a structured interview and questionnaire related to their medical conditions and underwent blood sampling for laboratory data and cytokines, including interleukin (IL)-6 and tumor necrosis factor (TNF)-α. Body mass index, dyspnea, the Eastern Cooperative Oncology Group performance status, and levels of albumin, blood urea nitrogen (BUN), total bilirubin, and C-reactive protein were significantly associated with fatigue. However, levels of the two proinflammatory cytokines, IL-6 and TNF-α, were not significantly correlated with the BFI-K score. In stepwise multiple linear regression, fatigue was related to elevated BUN (ß = 0.376, p = 0.002), severe pain intensity (ß = 0.349, p = 0.004), and impaired performance status (ß = 0.268, p = 0.027), but not related to levels of inflammatory cytokines. In conclusion, the diagnostic work-up and therapeutic plan for patients with cancer-related fatigue should include an evaluation of laboratory parameters, pain severity, and physical performance.

Proteomic analysis of tumor tissue in CT-26 implanted BALB/C mouse after treatment with ascorbic acid.

Tumor establishment and penetration consists of a series of complex processes involving multiple changes in gene expression and protein modification. Proteome changes of tumor tissue were investigated after intraperitoneal administration of a high concentration of ascorbic acid in BALB/C mice implanted with CT-26 cancer cells using two-dimensional gel electrophoresis and mass spectrometry. Eighteen protein spots were identified whose expression was different between control and ascorbic acid treatment groups. In particular, eukaryotic translation initiation factor 3 subunit 1, nucleophosmin, latexin, actin-related protein 2/3 complex subunit 5, M2-type pyruvate kinase, vimentin, tumor protein translationally-controlled 1, RAS oncogene family Ran, plastin 3 precursor, ATPase, Rho GDT dissociation inhibitor ß, and proteasome activator subunit 2 expression were quantitatively up-regulated. The increase in the level of these proteins was accompanied by an increase in mRNA level. The cytoskeleton protein actin, vimentin, and tumor protein translationally-controlled 1 showed quantitative expression profile differences. A change in actin cytoskeleton distribution, functionally relevant to the proteome result, was observed after treatment with ascorbic acid. These results suggest a previously undefined role of ascorbic acid in the regulation of cytoskeleton remodeling in tumor tissues.

Acquired lymphedema: Molecular contributors and future directions for developing intervention strategies.

Lymphedema is a debilitating chronic disease that mostly develops as an adverse reaction to cancer treatment modalities such as chemotherapy, surgery, and radiotherapy. Lymphedema also appears to be a deteriorating consequence of roundworm infections, as best represented by filariasis. According to its origin, lymphedema is classified as primary lymphedema and acquired lymphedema. The latter is an acquired condition that, hitherto, received a considerably low attention owing to the less number of fatal cases been reported. Notably, despite the low mortality rate in lymphedema, it has been widely reported to reduce the disease-free survival and thus the quality of life of affected patients. Hence, in this review, we focused on acquired lymphedema and orchestration of molecular interplays associated with either stimulation or inhibition of lymphedema development that were, in vast majority, clearly depicted in animal models with their specific and distinct technical approaches. We also discussed some recent progress made in phytochemical-based anti-lymphedema intervention strategies and the specific mechanisms underlying their anti-lymphedema properties. This review is crucial to understand not only the comprehensive aspects of the disease but also the future directions of the intervention strategies that can address the quality of life of affected patients rather than alleviating apparent symptoms only.

Production of human hyaluronidase in a plant-derived protein expression system: plant-based transient production of active human hyaluronidase.

Four types of human hyaluronidases (rHuHyal-1, -2, -3 and -4) were transiently expressed and purified from Nicotiana benthamiana, and their biochemical characteristics were analyzed. The recombinant HuHyals were expressed via agrobacteria-mediated infiltration and generated and expressed in terms of micrograms per 5 leaves of N. benthamiana. Expressed recombinant HuHyals were purified using a His(6) tagging system and Ni column chromatography, respectively, at pH 8.0, after which the purified rHuHyals were concentrated for additional biochemical analyses. The four types of rHuHyals were allowed to react with hyaluronic acids and chondroitin sulfates. The biochemical properties of rHuHyal-1 fit those of the commercially available Hyal, PH-20, which was extracted from animal testes under acidic conditions (pH 3.5). However, rHuHyal-1 evidenced activity levels 2 to 6-fold greater than the three other rHuHyals (rHuHyal-2, -3 and -4) at pH 3.5. However, only rHuHyal-4 exhibited chondroitinase activity with both 6-S-chondroitin sulfate (chondroitin sulfate C) and 4-S-chondroitin sulfate (chondroitin sulfate A) as standard substrates. The results of zymography demonstrated that recombinant HuHyal 1 was modified by glycosylation, but Escherichia coli Hyal was not. This result demonstrated that plant-based rHuHyal was functionally active and evidenced biochemical characteristics and post-translational protein modifications similar to those of animal testis-derived Hyal.

Construction of a new, objective prognostic score for terminally ill cancer patients: a multicenter study.

GOALS OF WORK: The goal of this study was to develop a new, objective prognostic score (OPS) for terminally ill cancer patients based on an integrated model that includes novel objective prognostic factors. MATERIALS AND METHODS: A multicenter study of 209 terminally ill cancer patients from six training hospitals in Korea were prospectively followed until death. The Cox proportional hazard model was used to adjust for the influence of clinical and laboratory variables on survival time. The OPS was calculated from the sum of partial scores obtained from seven significant predictors determined by the final model. The partial score was based on the hazard ratio of each predictor. The accuracy of the OPS was evaluated. MAIN RESULTS: The overall median survival was 26 days. On the multivariate analysis, reduced oral intake, resting dyspnea, low performance status, leukocytosis, elevated bilirubin, elevated creatinine, and elevated lactate dehydrogenase (LDH) were identified as poor prognostic factors. The range of OPS was from 0.0 to 7.0. For the above cutoff point of 3.0, the 3-week prediction sensitivity was 74.7%, the specificity was 76.5%, and the overall accuracy was 75.5%. CONCLUSIONS: We developed the new OPS, without clinician's survival estimates but including a new prognostic factor (LDH). This new instrument demonstrated accurate prediction of the 3-week survival. The OPS had acceptable accuracy in this study population (training set). Further validation is required on an independent population (testing set).

Enhanced Anticancer Effect of Adding Magnesium to Vitamin C Therapy: Inhibition of Hormetic Response by SVCT-2 Activation.

l-Ascorbic acid (vitamin C, AA) is known as an antioxidant, but at high concentrations, AA can kill cancer cells through a prooxidant property. Sodium-dependent vitamin C transporter family-2 (SVCT-2) determines the cellular uptake of AA, and the activity of SVCT-2 is directly related to the anticancer activity of AA. Cancer cells that showed high SVCT-2 expression levels were more sensitive to AA treatment than cancer cells with low SVCT-2 expression levels. Cells with low SVCT-2 expression showed a hormetic response to a low dose of AA. Magnesium ions, which are known to activate SVCT-2, could increase the Vmax value of SVCT-2, so we investigated whether providing magnesium supplements to cancer cells with low SVCT-2 expression that had shown a hormetic response to AA would elevate the Vmax value of SVCT-2, allowing more AA to accumulate. To evaluate the effects of magnesium on cancer cells, MgSO4 and MgCl2 were screened as magnesium supplements; both forms showed synergistic anticancer effects with AA. Taken together, the results of this study suggest that magnesium supplementation enhanced the anticancer effect of AA by inhibiting the hormetic response at a low dose. This study has also demonstrated that AA treatment with magnesium supplementation provided more effective anticancer therapy than AA treatment alone.

Proteomic analysis reveals upregulation of RKIP in S-180 implanted BALB/C mouse after treatment with ascorbic acid.

Tumor cells have an invasive and metastatic phenotype that is the main cause of death for cancer patients. Tumor establishment and penetration consists of a series of complex processes involving multiple changes in gene expression. In this study, intraperitoneal administration of a high concentration of ascorbic acid inhibited tumor establishment and increased survival of BALB/C mice implanted with S-180 sarcoma cancer cells. To identify proteins involved in the ascorbic acid-mediated inhibition of tumor progression, changes in the liver proteome associated with ascorbic acid treatment of BALB/C mice implanted with S-180 were investigated using two-dimensional gel electrophoresis and mass spectrometry. Eleven protein spots were identified whose expression was different between control and ascorbic acid treatment groups. In particular, Raf kinase inhibitory protein (RKIP) and annexin A5 expression were quantitatively up-regulated. The increase in RKIP protein level was detected in the tumor tissue and accompanied by an increase in mRNA level. Our results suggest a possibility that these proteins are related to the ascorbic acid-mediated suppression of tumor formation.

High dose concentration administration of ascorbic acid inhibits tumor growth in BALB/C mice implanted with sarcoma 180 cancer cells via the restriction of angiogenesis.

To test the carcinostatic effects of ascorbic acid, we challenged the mice of seven experimental groups with 1.7 x 10(-4) mol high dose concentration ascorbic acid after intraperitoneal administrating them with sarcoma S-180 cells. The survival rate was increased by 20% in the group that received high dose concentration ascorbic acid, compared to the control. The highest survival rate was observed in the group in which 1.7 x 10(-4) mol ascorbic acid had been continuously injected before and after the induction of cancer cells, rather than just after the induction of cancer cells. The expression of three angiogenesis-related genes was inhibited by 0.3 times in bFGF, 7 times in VEGF and 4 times in MMP2 of the groups with higher survival rates. Biopsy Results, gene expression studies, and wound healing analysis in vivo and in vitro suggested that the carcinostatic effect induced by high dose concentration ascorbic acid occurred through inhibition of angiogenesis.

Yeast (1 → 3)-(1 → 6)-ß-d-glucan alleviates immunosuppression in gemcitabine-treated mice.

Gemcitabine (2'-deoxy-2',2'-difluorocytidine, dFdC) is one of the most effective chemotherapy drugs commonly used for treatment of various tumors. Despite its significant anticancer effects, some adverse effects create obstacles to treatment. The main toxicity of gemcitabine is myelosuppression, which not only reduces patient quality of life, but also hinders further anticancer treatment. In this respect, immunotherapy can address these drawbacks because of its ability to enhance the patient's immune system. To improve immune system function, yeast-derived ß-glucans, which are well-known biologic response modifiers, were administered to gemcitabine-treated mice. The in vivo experiment revealed that orally administered yeast (1 → 3)-(1 → 6)-ß-d-glucan effectively alleviated myelosuppression associated with gemcitabine-induced pancytopenia. Moreover, analysis of myelopoiesis-related cytokine expression through real-time PCR demonstrated that ß-glucan treatment significantly upregulated hematopoietic responses in gemcitabine-treated mice. Furthermore, orally administered ß-glucan significantly induced the expression of IFN-γ and IL-2 in splenocytes of gemcitabine-treated mice. It also restored the cytotoxicity of splenocytes against YAC-1 in gemcitabine-treated mice and displayed a positive effect on gemcitabine-damaged bone marrow tissue. In conclusion, yeast ß-glucans have the potential to be used as adjuvants for alleviating chemotherapy-induced immunosuppression in patients.

Hormetic dose response to L-ascorbic acid as an anti-cancer drug in colorectal cancer cell lines according to SVCT-2 expression.

L-Ascorbic acid (vitamin C, AA) exhibits anti-cancer effects with high-dose treatment through the generation of reactive oxygen species (ROS) and selective damage to cancer cells. The anti-cancer effects of L-ascorbic acid are determined by sodium-dependent vitamin C transporter 2 (SVCT-2), a transporter of L-ascorbic acid. In this study, we demonstrate that L-ascorbic acid treatment showed efficient anti-cancer activity in cell lines with high expression levels of SVCT-2 for a gradient concentration of L-ascorbic acid from 10 µM -2 mM. However, in low SVCT-2 expressing cell lines, high-dose L-ascorbic acid (>1 mM) showed anti-cancer effects but low-dose (<10 µM) treatment induced cell proliferation. Such conflicting results that depend on the concentration are called a hormetic dose response. A hormetic dose response to low-dose L-ascorbic acid was also observed in high SVCT-2 expressing cell lines in the presence of a SVCT family inhibitor. Insufficient uptake of L-ascorbic acid in low SVCT-2 expressing cancer cell lines cannot generate sufficient ROS to kill cancer cells, resulting in the hormetic response. Molecular analysis confirmed the increased expression of cancer proliferation markers in the hormetic dose response. These results suggest that L-ascorbic exhibits a biphasic effect in cancer cells depending on SVCT-2 expression.

Glutathione alleviated peripheral neuropathy in oxaliplatin-treated mice by removing aluminum from dorsal root ganglia.

Oxaliplatin, a platinum-based anti-cancer drug, induces peripheral neuropathy as a side effect and causes cold hyperalgesia in cancer patients receiving anti-cancer chemotherapy. In oxaliplatin-treated mice, aluminum was accumulated in the dorsal root ganglia (DRG), and accumulated aluminum in DRG or other organs aggravated oxaliplatin-induced neuropathic pain. To investigate whether aluminum oxalate, which is the compound of aluminum and oxaliplatin, might be the peripheral neuropathy inducer, the withdrawal responses of mice to coldness, the expression of transient receptor potential ankyrin 1 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays in DRG were analyzed in mice administered with aluminum oxalate. In addition, the concentrations of aluminum in aluminum oxalate-treated mice were significantly increased compared to those of mice treated with aluminum chloride. To alleviate neuropathic pain, glutathione (GSH), known as an antioxidant and a metal chelator, was injected into oxaliplatin-treated mice. The concentrations of aluminum in the DRG were decreased by the chelation action of GSH. Taken together, behavioral and molecular analyses also supported that aluminum accumulation on the DRG might be a factor for neuropathic pain. This result also suggested that the aluminum chelation by GSH can provide an alleviatory remedy of neuropathic pain for cancer patients with oxaliplatin-induced neuropathic pain.

Therapeutic effects of hyaluronidase on acquired lymphedema using a newly developed mouse limb model.

Acquired lymphedema is one of the most dreaded side effects of cancer treatment, such as surgical treatment or irradiation. However, due to the lack of appropriate animal models, there is no effective therapeutic method to cure acquired lymphedema. To develop a reproducible acquired lymphedema animal model, we devised a mouse hind limb model by removing a superficial inguinal lymph node, a popliteal lymph node, a deep inguinal lymph node, and the femoral lymphatic vessel. We measured the volume of lymphedematous leg and observed the change in level of hyaluronic acid (HA) and lymphangiogenic factors after injecting hyaluronidase. Our model showed the distinguishable swelling and the reliable symptoms compared to previously reported models. In the lymphedematous regions of our model, we confirmed that HA, a major component of extracellular matrix, accumulated to higher levels than in a normal mouse. This lymphedema volume was rapidly reduced by treating hyaluronidase. Following hyaluronidase injection, the lymphedematous region of our model resembled a normal hind limb. Our findings indicated that hyaluronidase promoted lymphangiogenesis on the lymphedematous limb. Based on hyaluronidase treatment in the lymphedematous region, this could potentially be a new therapeutic approach for acquired lymphedema mediated through the modification of the size of HA fragments. Impact statement In this manuscript, the essence of the work described in this manuscript involves the development of (1) a mouse limb model showing acquired lymphedema and (2) a potent therapeutic treatment using hyaluronidase to remedy acquired lymphedema in our model. In order to develop a reproducible acquired lymphedema animal model that reflects the most common symptoms experienced by lymphedema patients, we devised a mouse hind limb model by removing lymph nodes and lymphatics. Our model showed the distinguishable swelling and the reliable symptoms compared to previously reported models. In the lymphedematous regions of our model, we confirmed that hyaluronic acid (HA) accumulated to higher levels than in a normal mouse. This lymphedema volume was rapidly reduced by treating the lymphedematous leg with hyaluronidase, which also degraded high molecular weight HA to low molecular weight HA. Immunohistochemical analysis, quantitative real-time PCR analysis and lymphangioscintigraphy showed that hyaluronidase enhanced lymphangiogenesis in the lymphedematous limb.

Hydrolysis of Hyaluronic Acid in Lymphedematous Tissue Alleviates Fibrogenesis via TH1 Cell-Mediated Cytokine Expression.

Although surgery and radiation are beneficial for treating cancer, they can also lead to malfunctions of the lymphatic system such as secondary lymphedema. This abnormality of the lymphatic system is characterized by severe swelling, adipogenesis, inflammation, and fibrosis in the lymphedematous region. Moreover, the proliferation of fibrotic tissue in the lymphedematous region generates edema that is no longer spontaneously reversible. No treatment for fibrosis has been validated in patients with lymphedema. In our efforts to develop a therapeutic agent for lymphedema fibrosis, we used a newly established mouse hind limb model. Previous studies have demonstrated that hyaluronic acid accumulates in the lymphedematous region. Thus, we challenged mice with of hyaluronidase (HYAL), with the aim of reducing fibrogenesis. After subcutaneous injections in the lymphedematous mouse leg every two days, the volume of lymphedema had reduced significantly by 7 days post-operation. Histochemical analysis indicated that collagen accumulation and myofibroblast differentiation were decreased in epidermal tissues after HYAL injection. Moreover, it was associated with upregulation of interferon-gamma, increased numbers of Th1 cells, and downregulation of interleukin-4 and interleukin-6 in the lymphedematous region and spleen. These results indicate that hydrolysis of hyaluronic acid can boost an anti-fibrotic immune response in the mouse lymphedema model.