Abstract
Natural products play an important role in medicine and human healthcare and, as a result, drugs that are derived from natural resources are increasingly being used for the treatment of various diseases, including cancer. Vietlife-Antican (VLA) is a novel combination of curcumin solid lipid nanoparticles, ginger oleoresin solid lipid nanoparticles, and rutin solid lipid nanoparticles, which all possess low toxicity and enhanced bioavailabilities. To clarify the antitumor effects of this product, cytotoxic tests of VLA were conducted in various cancer cell lines in vitro and in an in vivo tumorized mouse model induced by Lewis lung carcinoma (LLC) cells. VLA expressed some cytotoxicity against breast cancer cells (MCF7), hepatocarcinoma cells (HepG2), lung cancer cells (SK-LU-1), colorectal adenocarcinoma cells (SW480), and human leukemia cells (HL-60). Additionally, LLC tumor-bearing mice treated with VLA (600 mg/kg body weight [bw]) exhibited significant reductions in tumor growth (P < 0.05), a longer mean survival time (34.05 days), and improved liver function. Furthermore, a combination of VLA (200 mg/kg bw) and cisplatin strongly suppressed tumor development and increased mean survival time (P < 0.05). Interestingly, cotreatment with VLA and cisplatin also induced the serum production of interleukin (IL)-2 and IL-10, which are important antitumor and anti-inflammatory cytokines, respectively, in tumorized mice. Taken together, the present results suggest that VLA may be a very promising and efficient phytochemical product that can support cancer treatment or therapy.
Keywords
In recent years, life expectancy has increased in conjunction with the development of science and technology. However, demographic transitions that involve the growth of a population as well as an increase in the number of elderly people have led to a shift in epidemiologies that result in disease and death in many countries, especially developing countries. 1 This transformation is evident in the increased incidence of chronic health issues such as cardiovascular diseases, cancer, diabetes, and chronic respiratory diseases.
Of these diseases, cancer has a high mortality rate that included approximately 9.6 million cases in 2018 and, potentially, will include 14.6 million cases in 2035 (World Health Organization [WHO], 2018). Although treatments such as chemotherapy, radiotherapy, surgery, and effective immunotherapies can enhance the longevity of patients, these therapeutic modalities are associated with adverse side effects that seriously impact patient’s quality of life or even the efficiency of a drug. Additionally, the total cost of cancer treatment increases every year. Therefore, there is a need for novel drugs with few side effects, effective characteristics, and a relatively low cost. As a result, recent studies have targeted natural active ingredients, especially plant-derived components. A category of natural active substances that includes paclitaxel, vincristine, and homoharringtonine has already been approved by the FDA for use as cancer treatments 2 and, furthermore, hundreds of other natural active compounds have been researched as possible treatments for cancer.
Curcumin is an active ingredient isolated from turmeric that is being clinically tested as a cancer therapeutic due to its anti-inflammatory and antioxidant capabilities, and tumor-related toxic effects on a variety of cancer cell lines. 3 -5 However, the poor absorption of curcumin limits its bioavailability and thus new modalities such as nanomaterials (eg, liposomes) or combinations of active ingredients are being assessed to determine if they can address this issue. The present study demonstrated the promising in vivo anticancer capabilities of the Vietlife-Antican (VLA) product, which is composed of curcumin solid lipid nanoparticles, ginger oleoresin solid lipid nanoparticles, and rutin solid lipid nanoparticles; these nanoparticles were formulated into a VLA softgel pill (confidential formula from Vietlife Healthcare).
Prior to performing the in vivo tests, the cytotoxic effects of VLA were assessed in several cancer cell lines. VLA displayed slight cytotoxic potential in all of the studied cell lines, including breast cancer cells (MCF7), hepatocarcinoma cells (HepG2), lung cancer cells (SK-LU-1), colorectal adenocarcinoma cells (SW480), and human leukemia cells (HL-60). VLA was more effective on SW480 and HL-60 cells, which have similar IC50 values ranging from 181.64 to 187.86 µg/mL (Figure 1), whereas it was less effective on MCF-7 and HepG2 cells, which have IC50 values of 212.00 and 227.05 µg/mL, respectively. In addition to the cytotoxic effects of VLA on cancer cells, its effects on noncancerous cells were determined in kidney cells (HEK-293). The IC50 of VLA on HEK-293 cells (249.18 µg/mL) was higher than those of the studied cancerous cell lines, which indicates that VLA might be less toxic on normal cells than on some cancerous cell lines.
The IC50 values of Vietlife-Antican (VLA) obtained from cytotoxic tests on several cancerous cell lines including breast cancer cells (MCF7), hepatocarcinoma cells (HepG2), lung cancer cells (SK-LU-1), colorectal adenocarcinoma (SW480), human leukemia cells (HL-60), and noncancerous cell line (HEK-293). Data were shown as mean ± SD. *P < 0.05, **P < 0.01 compared with the noncancerous cells were considered as statistically significant.
Due to its potential cytotoxic activities, the antitumor effects of VLA were also tested in Lewis lung carcinoma (LLC tumor-bearing mice. Previous acute toxic studies have shown that VLA induced no effects on survival, clinical observations, body weight (bw) or food, or water consumption in mice up to 16.20 g/kg bw (oral administration) dose (Supplementary Table Appendix 1). In addition, from subchronic toxic tests, VLA did not significantly change the behavior, bw, or hematological, biochemical aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatine parameters at doses of 200, 400, and 600 mg/kg bw for 45 days (data not shown, P>0.05; Supplementary Table Appendix 2); hence doses of 200 and 600 mg/kg bw were selected for the antitumor study. The tumorized mice were randomly divided into the following groups: negative control, VLA treatment at 200 mg/kg bw, VLA treatment at 600 mg/kg bw, cisplatin alone at 2 mg/kg bw, and a combination of cisplatin at 2 mg/kg bw and VLA at 200 mg/kg bw. The tumor volume increased quickly in the control group (Figure 2a), and VLA at 200 mg/kg did not significantly reduce the tumor volume in the initial stages but resulted in the mild suppression of tumor growth at later stages. In contrast, tumor volume in the VLA at 600 mg/kg group was significantly lower in both the early and late stages (P < 0.01). At the end of the study, VLA at 600 mg/kg inhibited tumor growth by 29.57% compared to the control group and the tumor weight was also lower than that of the control group. Additionally, VLA at this dose exhibited a significantly longer mean survival time which was 34.05 days compared with that of the control group (P<0.05, Figure 2d). These results indicate that the higher dose of VLA effectively suppressed the progress of LLC tumors.
Tumor inhibition efficacy of Vietlife-Antican (VLA) and combination of VLA + cisplatin at different time points. Lewis lung carcinoma tumor-bearing mice were divided into different groups including control group, which received normal saline, VLA-treated groups (200, 600 mg/kg body weight [bw]), cisplatin (2 mg/kg bw, IV) treated group, and combination of VLA (200 mg/kg bw) with cisplatin (2 mg/kg bw, IV); (a) the growth of tumor volume at different time points; (b) tumor weight on day 28; (c) effect of VLA on bw at the start and the end of study; (d) mean survival time of VLA-treated tumor mice. Data were presented as mean ± SE. *P < 0.05, **P < 0.01 compared with the control group were considered as statistically significant.
In the present study, one group of tumorized mice was treated with cisplatin (2 mg/kg, IV) on day 5 and day 12 after LLC implantation as a reference control and there was a slight reduction in tumor volume on day 28 of cisplatin treatment. Cisplatin is a well-known FDA-approved chemotherapeutic drug that was developed from platinum. Due to its potential for resisting various types of cancers, including sarcomas, both nonsmall and small-cell lung cancers and cancers of the soft tissue, bones, muscles, and blood vessels, 6,7 cisplatin has been widely used as a cancer treatment. However, the side effects associated with cisplatin include neurotoxicity, renal toxicity, and/or bone marrow suppression as well as chemotherapy-resistant reductions in the effectiveness of cisplatin and decreased quality of life in patients. 8 A combination of cisplatin with natural compounds is thought to reduce side effects as well as improve treatment efficacy. For example, a combination of cisplatin and curcumin effectively treats bladder cancer in nude mice bearing 253J-Bv cell xenografts. 9 In the present study, treatment with VLA at 200 mg/kg alone did not clearly inhibit tumor growth but a combination of oral VLA at 200 mg/kg and IV cisplatin at 2 mg/kg significantly suppressed tumor development by up to 49.67% compared to cisplatin alone (22.90%) or VLA alone on day 28. The ability of this combination to inhibit tumor activity was also confirmed by lower tumor weights compared to the control and cisplatin-alone groups (Figure 2b; P < 0.01). Additionally, there were no significant changes in bw in the experimental groups compared to the control group, which is indicative of the low toxicity of VLA (Figure 2c).
A variety of treatment-related side effects, including anemia and immunosuppression, can occur during the cancer therapy process and result in a lack of drug specificity. Therefore, the hematological parameters of the experimental groups were evaluated in the present study. VLA treatment did not induce anemia in any of the groups (Table 1), which was evidenced by the similar red blood cell (RBC) counts, hemoglobin (Hb) levels, and/or hematocrit numbers of the treatment groups and the control group. Treatment with VLA also slightly reduced the number of leukocytes compared to the control group but these differences were not significant (P > 0.05). Several other biochemical parameters were also evaluated. Because the liver and kidney are involved in detoxification and excretion, they are easily affected by exogenous drugs and the side effects of many chemotherapeutics are toxic to these organs. In the present mouse model, the growth of LLC tumors induced liver toxicity that was evident in very high AST levels (1007.89 UI/L; Table 1). Chen et al. 10 reported increased AST release in cases of ischemia, hypoxia, damaged cell membrane integrity, functional damage, and abdominal injury. AST levels have been correlated with the prognoses of several cancers but the administration of VLA at 600 mg/kg alone or a combination of VLA at 200 mg/kg and cisplatin significantly repressed the production of AST (P < 0.05). There were no significant changes in another liver enzyme, ALT, in any of the treated groups. Taken together, these results indicate that VLA may protect hepatocytes against the toxicity induced by LLC tumors.
The Effects of VLA Alone or in Combination with Cisplatin on the Hematological Parameters of Lewis Lung Carcinoma Tumor-Bearing Mice.
ALT, alanine aminotransferase; AST, aspartate aminotransferase; Hb, hemoglobin; MCV, mean corpuscular (cell) volume; MCHC, mean corpuscular hemoglobin concentration; RBC, red blood cell; VLA, Vietlife-Antican.
* P < 0.05 compared to the control.
Because high doses of VLA alone and VLA plus cisplatin were significantly protective against tumor development in the present study, further analyses of the serum production of the antitumor cytokine interleukin (IL)-2 and the anti-inflammatory cytokine IL-10 were conducted in the tumorized mice. As shown in Figure 3, although the administration of VLA at 600 mg/kg induced slight alterations in the productions of IL-2 (Figure 3a) and IL-10 (Figure 3b), these changes were not statistically significant (P > 0.05). Interestingly, the serum levels of IL-2 and IL-10 in the tumorized mice significantly increased when they were treated with a combination of VLA and cisplatin. The functions of IL-2 and IL-10 in cancer have been previously described. For example, IL-2 and IL-10 operate as immunomodulatory factors that control the proliferation levels and actions of lymphocytes, macrophages, and natural killer cells. Additionally, IL-2 and IL-10 exert synergistic effects via the enhancement of CD8+ T cell functions by improving their survival, proliferation, and cytotoxicity of tumor antigen-specific CD8+ T cells. 11,12 Therefore, inducing the productions of IL-2 and IL-10 may result in an enhanced immunological reaction in cancer patients that can help prolong survival time.
Effect of Vietlife-Antican (VLA) on the production of interleukin (IL)-2 (a) and IL-10 (b) in tumor mice serum. The data were collected from at least 5 mice per group. *P < 0.05 compared with the control group was considered as statistically significant.
Previous studies have indicated that curcumin exerts anticancer activities in both in vitro and in vivo models 13 and that it is effective against melanoma as well as head and neck, breast, colon, pancreatic, prostate, and ovarian cancers. 5,14 Curcumin impacts cancer via several mechanisms including suppressing the NF-κB activation, controlling the cell cycle, and inducing cell death through the stimulation of cell apoptosis and autophagy. Curcumin also induces anti-inflammatory and antioxidant activities via the inhibition of lipid peroxidation and downregulation of iNOS activity. 15 However, curcumin is a hydrophobic polyphenol, which results in weak oral bioavailability. The production of curcumin nanoparticles in VLA is aimed at resolving this issue by improving its delivery and enhancing its concentration in tumor cells. 13
The components of VLA also include ginger and rutin at a nanoscale level. Ginger is another important food in traditional medicine that is a well-known anticancer drug. Phenolic compounds of ginger express strong antioxidative and anti-inflammatory actions and suppress the initiation and promotion of carcinogenesis. 16 Ginger is also known to exert antiproliferative activities in ovarian cancer 17 and pancreatic cancer 18 cells. Rutin is an active compound that inhibits platelet aggregation and possesses anti-inflammatory and antioxidant capabilities. This compound contributes to decreases in the amount of precancerous lesions and the induction of apoptosis in large intestine cancer and thus has the potential to contribute to treatments for other types of cancer. 19 The promotion of cancer is associated with a variety of processes, including the proliferation, migration, and metastasis of cancer cells as well as inflammation and hypoxia and, together, these elements contribute to the seriousness of the disease status. The incorporation of nanocurcumin, nanoginger, and nanorutin may provide novel pathways for the suppression of tumor growth. The present study confirmed the cytotoxicity of VLA against several cancer cell types that have IC50 values < 250 µg/mL. Additionally, at higher doses, VLA displayed promising in vivo antitumor activities through the inhibition of tumor growth, prolonging survival time, and protecting the liver from tumor-induced damage.
Interestingly, all components of VLA are natural and have a low toxicity, which contribute to the safety of the VLA product. Additionally, the antioxidant and anti-inflammatory activities of the VLA components may help to reduce the varied side effects of other chemotherapies and radiotherapies. Thus VLA is an ideal solution for combining and improving the effectiveness of many chemotherapeutics. In the present study, VLA was also administrated in combination with cisplatin to investigate its antitumor effectiveness in an LLC tumor-bearing mouse model. The antioxidant activities of VLA helped to reduce the oxidative stress induced by cisplatin. Thus, VLA might also stimulate activity in different pathways to inhibit tumor growth and prolong the survival time of tumorized mice. The combination of VLA and cisplatin significantly inhibited tumor growth compared to the control group and cisplatin alone groups (P < 0.05).
In conclusion, the present study found that VLA exhibited promising antitumor activities both in vitro against various cancer cell lines and in vivo in tumor-bearing mice. Treatment with VLA at 600 mg/kg bw significantly suppressed tumor growth while the administration of VLA at a low dose strongly enhanced the antitumor effectiveness of cisplatin. Therefore, VLA may be a reliable approach for reducing cancer growth, increasing survival time, and improving the quality of life in cancer patients.
Experimental
Material
VLA (softgel capsule, 750 mg) provided by Vietlife Healthcare Corp. with active ingredients including nanocurcumin 50 mg (curcuminoid 22%), 25 mg nanoginger, and 0.5 mg nanorutin.
Chemicals
Eagle’s minimum essential medium (EMEM), trypsin-EDTA, and gentamicin were obtained from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), MEM nonessential amino acids (NAA), and sulforhodamine B (SRB) were purchased from Sigma Chemicals (St. Louis, MO, USA).
Cell Lines and Cell Culture
LLC, SK-LU-1, MCF7, HepG2, SW480, HL-60, and HEK-293 were kindly provided by Prof J. Maier, Milan University, Italia, and Prof J.M. Pezzuto, Rutgers University, USA. HL-60 was maintained in RPMI medium with 10% FBS and gentamicin (50 µg/mL). Other cell lines were cultured in EMEM containing 1% NAA, gentamicin, and 10% FBS in the incubator at 37°C and 5% CO2. Cells were subcultured every 2 days.
Cytotoxic Activity
The cytotoxic activity of the sample was determined by SRB assay. Briefly, cells were seeded in the 96-well plates at a concentration of 3 × 104 cells/mL and treated with different concentrations of sample for 48 hours in the incubator at 37°C and 5% CO2. After that, cells were fixed with TCA, washed under tap water and stained with SRB 0.4% for 30 minutes. The protein-binding SRB dye, which is related with cell viability, was dissolved in Tris buffer. The optical density was determined at 540 nm by ELISA reader (Tecan GENios Plate Reader). IC50 values were calculated based on the percent inhibition of cell growth using Tablecurve 2Dv4 software.
Animal and Antitumor Activity
A total of 100 healthy BALB/c mice (in range of 25-30 g) were housed in plastic cages in the Institute of Biotechnology, Vietnam Academy of Science and Technology (VAST), with a 12 hours light/dark cycle at 23°C to 25°C. Experiments were performed in accordance with Vietnamese Ethical Laws; European Communities Council Directives of November 24, 1986 (86/609/EEC) guidelines; and approval from Scientific Council of Institute of Biotechnology, VAST, for the care and use of laboratory animals. Mice were given control food and ad libitum water. In order to induce tumors, LLC cells were SC implanted to the right leg of mice. After 5 days, LLC tumor-bearing mice were divided into 10 groups, in which groups 1 and 2 served as control; groups 3 and 4 were treated with VLA at a dose of 200 mg/kg bw for continuous 28 days; groups 5 and 6 were treated with 600 mg/kg bw of VLA for continuous 28 days; cisplatin, served as reference control, was given intravenous to the tail vein of mice in group 7 and 8 at day 5 and day 12 after LLC injection. To study the effect of VLA on tumor growth when combined with cisplatin, mice in groups 9 and 10 received 200 mg/kg bw of VLA for continuous 28 days and were IV injected 2 mg/kg bw of cisplatin at days 5 and 12 after LLC injection. In the study period, tumor size of all groups was measured every 7 days by caliper and tumor volume ( V) was calculated by the formula: V = (a 2 × b)/2 in which a and b are the smallest diameter and the largest diameter, respectively.
On day 28 of treatment, blood of mice in groups 1, 3, 5, 7, and 9 was collected to determine hematological such as RBC, white blood cells, Hb, mean corpuscular (cell) volume, mean corpuscular Hb concentration by using a blood automatic analyzer (AU680, Beckman coulter) and chemical parameters (serum AST, ALT, and creatine). The levels of IL-2 and IL-10 in serum were also investigated by using commercial ELISA kits (Biovision, Chester Springs, PA, USA), following the manufacturer’s protocols. After that, mice in these groups were anesthetized and tumors separated to measure the tumor weight. Mice in other groups (2, 4, 6, 8, 10) were continuously monitored and the survival time assessed.
Statistical Analysis
Data were collected and expressed as mean ± SEM and analyzed by GraphPad Prism 4.0 (GraphPad Software, Inc., San Diego CA). Student’s t-test and one-way ANOVA were used for significant statistics. P < 0.05 was considered statistically significant in all comparisons.
Supplemental Material
Supplementary Tables - Supplemental material for Potential Anticancer Activities of a Combination of Curcumin, Ginger Oleoresin, and Rutin Solid Lipid Nanoparticles (Vietlife-Antican) in LLC Tumor-Bearing Mice
Supplemental material, Supplementary Tables, for Potential Anticancer Activities of a Combination of Curcumin, Ginger Oleoresin, and Rutin Solid Lipid Nanoparticles (Vietlife-Antican) in LLC Tumor-Bearing Mice by Do Thi Thao, Nguyen Thi Nga, Nguyen Anh Van, and Kieu Dinh Hung in Natural Product Communications
Footnotes
Acknowledgment
The authors are grateful to Prof Dr Nguyen Duc Nghia, the Institute of Chemistry, VAST, for his great support with all nanomaterials that were used in the VLA ingredients.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) declared no financial support for the research, authorship, and/or publication of this article.
References
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
