INTRODUCTION:

Cancer is one of the most widely discussed issues in the world, owing to the high number of deaths and the economic burden¹. More than 19.2 million new cancer cases were recorded in 2020, accounting for approximately 10 million deaths worldwide². Liver cancer is one of the most common cancers, accounting for the sixth-highest number of new cases and the third-highest number of deaths². Hepatoblastoma is the most common hepatic malignancy in pediatric patients, accounting for approximately 80% of all cases³.

In the late stages of liver cancer, 80 percent of patients are unable to undergo surgery⁴. The 10-year survival rate for patients who have had liver cancer surgery is only 60% for children and 34% for minors³. Sorafenib is a well-known targeted therapy for the treatment of liver cancer^5,6. However, due to concerns about side effects and drug resistance, drug combinations have been proposed to improve treatment efficacy^7-10. Many studies have been conducted to test the combination of Sorafenib and cytostatic agents, which have proven effective in the treatment of liver tumors (bevacizumab, cisplatin, irinotecan, etc.)^11,12. Plant-derived compounds have been validated as a source of anticancer agents^13-19. The presence of natural compounds with high content, such as terpenoids and curcuminoid, aids in the formation of bioactive compounds in Curcuma aromatica^20-24. Numerous studies have shown that this plant has antioxidant and antitumor properties, including anti-liver cancer ability^24-28. Herbs containing curcumin have recently been targeted as sources of supportive factors in cancer treatment²⁹. As a result, this study was conducted to assess the combined effect of C. aromatica and Sorafenib in the treatment of liver cancer.

MATERIALS AND METHODS:

Plant materials and sample extraction preparation:

Curcuma aromatica was collected in the mountains of Vietnam's An Giang province. The plant was identified by observing distinct morphological characteristics and confirmed by molecular biology techniques using the marker gene trn S-trnf M.

The rhizome puree was moistened in 96 percent ethanol for two hours before macerating overnight in 96 percent ethanol. Evaporating the extract yielded the crude extract, which was then dissolved in DMSO for analysis.

Drugs preparation:

Sorafenib (Sigma-Aldrich) was dissolved in DMSO for drugs combined evaluation.

Cell culture preparation:

For testing, the human hepatoblastoma cell line Hep J5 (ATCC, USA) was used. Cells grow as a monolayer in DMEM (Dulbecco's Modified Eagle Medium, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (ThermoFisher Scientific) and 1% penicillin-streptomycin (Sigma-Aldrich) at 37°C in a humidified incubator with 5% CO₂. After 2-3 days, the cell was sub-cultured.

Cytotoxic assay:

The extract's lethality was investigated using the SRB method³⁰. Cells were seeded into Elisa plates at a density of 3000 cells per well and treated at various concentrations. After 24 hours of exposure, the absorbance at 595nm was measured to determine the half-maximal inhibitory concentration (IC₅₀).

Clonogenic assay:

The extract's inhibitory capacity was investigated by assessing its clonogenic ability³¹. Cells were seeded at a density of 200-300 cells per well in a six-well culture plate. After 24 hours of growth, the cell was treated for 1 - 3 weeks until cell cluster formation. The wells were washed twice with PBS and then dyed with H&E (Merck) for observation. The cell clustering rate was calculated as a percentage of the treatment and control groups.

Migration assay:

The migration assays were used to examine cell migration during treatment³². In a 6-well plate, cells were seeded and cultured as a monolayer in DMEM supplemented with 10% FBS and 1% Penicillin-Streptomycin until 80 - 90% of the well bottom surface was covered. Cross-cutting with a 10mL pipette tip resulted in a free cell gap. For cell debris removal, the well was washed twice with PBS. Following incision, the cell grew in medium with or without treatment supplement at 37°C and 5% CO₂. Cell growth was recoded at 0 hours, 24, and 48 hours.

Data analysis:

The tests were performed at least three times. Data were calculate using SPSS version 16.0. Significant differences were determined by p-value of 0.05(*) and 0.01(**). Data were presented as mean±standard error of mean. The drug interaction was investigated by CalcuSyn software (www.combosyn.com) and determined the combination index (CI).

RESULTS AND DISCUSSION:

Sorafenib was evaluated and shown to be effective in treating patients with liver cancer in the late 2000s^6,33. Sorafenib, which was previously approved for advanced renal cell carcinoma treatment in 2005 and metastatic differentiated thyroid cancer treatment in 2013, was approved by the FDA in 2008 for use in the treatment of hepatocellular carcinoma^33-36. Sorafenib is a multi-target kinase inhibitor that inhibits multiple signaling pathways, including the Raf/MEK/ERK pathway⁶, the VEGFR-2 and 3 receptors, FLT-3, and c-KIT³⁷. Sorafenib's therapeutic use has also been linked to side effects such as diarrhea, hypertension, and skin toxicity⁸. As a result, combining Sorafenib with other therapeutic agents is required to avoid unwanted side effects³⁸.

Figure 1. The effect of the combination on Hep J5 cell (A); The results of isobologram analysis and CI distribution (B)

The interaction of Sorafenib and C. aromatica extract was studied using a cytotoxic assay on the liver cancer cell line Hep J5. The IC₅₀ was calculated using a non-linear regression equation. There was a significant difference in the effect of Sorafenib on Hep J5 in the presence of C. aromatica extract at different concentrations (Figure 1A). At the same Sorafenib concentration, the more C. aromatica extract supplemented, the higher the cell death rate, with a statistically significant difference. To assess the relative potency of the combination, an isobologram model integrated into the CalcuSyn software was used. The CI value, which is calculated as the sum of the ratios of individual effect and combined effect at the concentration of IC₅₀ values for the two ingredients, determines whether the combination is synergistic or antagonistic³⁹. Schematic isobologram and CI distribution chart were shown in figure 2, all the data points were located in synergistic area below the additively line. The drugs interactions are considered as synergistic if the CI value is less than 0.8 (<0.1:Very strong synergism; 0.1-0.3: strong synergism; 0.3-0.7: synergism and 0.7-0.85: moderate synergism) and antagonistic if the CI value is greater than 1 (>10:Very strong antagonism; 3.30-10: strong antagonism; 1.45-3.30: antagonism and 1.20-1.45: moderate antagonism), the CI value that fell in the range of 0.8-1.2 indicates the combination is additive⁴⁰.

Table 1. Summary of CI values from cytotoxic assay of the combination

*Dose C. aromatica* (µg/mL)**	Dose Sorafenib (µM)	Effect	CI	Description
125.00	1.00	0.45	0.75848	moderate synergism
125.00	2.00	0.57	0.67019	synergism
125.00	5.00	0.66	0.80985	moderate synergism
250.00	1.00	0.65	0.71274	moderate synergism
250.00	2.00	0.74	0.59562	synergism
250.00	5.00	0.88	0.39537	synergism

Table 1 shows the CI of the combination after calculation and statistics. The results showed that combining Sorafenib and C. aromatica extract had a synergistic effect on the cell line Hep J5, which was a critical principle in the current clinical drug combination.

The combination of Sorafenib and C. aromatica extract at concentrations lower than the IC₅₀ for each agent was then used to assess Hep J5 clonogenic ability and cell migration. The percentage of Hep J5 cell clonogenic formation under combined treatment was obtained at the much lower value (19%) than both of the individual treatment of Sorafenib (65%) and C. aromatica extract (75%) (Figure 2). The difference in colony formation between the three experimental groups was statistically significant, with the combined group having a p-value less than 0.01. When compared to MTT and SBR assays, the clonogenic assay has been shown to be the most effective in investigating drug sensitivity⁴¹. The cancer cell's limited colony-forming capacity reflected its therapeutic sensitivity to treatment^42,43.

Figure 2. Hep J5 colony formation under the treatment of Sorafenib (2µM), C. aromatica (125µg/mL) extract and the combination observed under microscope (A) and statistics on the histogram (B)

Hep J5 cell migration was specified as 10% under the influence of the combination and 60% and 55% for exposure alone with Sorafenib and C. aromatica extract, respectively (Figure 3). This result was similar to what was observed in the clonogenic assay, with a statistically significant difference between the three treatment groups. The study's findings revealed that the cellular response to treatment was time and dose-dependent. The combination's effect is manifested by the ability to inhibit cell proliferation, migration, and colony formation.

Figure 3. Migrating ability of Hep J5 cells before and after Sorafenib, C. aromatica and combination treatment observed under the microscope (A) and statistics on the histogram (B)

C. aromatica extract was shown to have anticancer properties by inhibiting cell proliferation and angiogenesis via VEGF in Ehrlich ascites carcinoma and inducing G2/M phase arrest in colon carcinoma^44,45. Curcumin is a key component in the development of bioactivity in curcuma species⁴⁶. The total curcumin content of C. aromatica was found to be quite high, at 6.07mg/100mg d.w.⁴⁷. The presence of two phenyl rings in the structure, as well as substituent groups such as hydroxyl and methoxyl, makes curcumin and its derivatives susceptible to interfering with cellular activities⁴⁸. Curcumin has been reported to be a potent inducer of cancer cell apoptosis via a variety of pathways, including regulation of the activity of the B-cl2 protein family, Bax, apoptogenic protein (cytochrome C, AIF), or inhibition of the PI3K/Akt/mTOR pathway, among others^49-52. Autophagy was shown to be initiated by the action of curcumin⁵¹. Curcumin has been shown to work synergistically with other compounds to inhibit cancer growth, such as piperine conjugation on Dalton lymphoma and resveratrol on Hepatocellular carcinoma^53,54. Previous research has shown that curcumin can be combined with Celecoxib for breast cancer treatment, Paclitaxel for effect enhancement, Doxorubicin, Cisplatin, and other chemotherapy drugs^55-57. In this study, the evaluated synergistic effect of Sorafenib and C. aromatica extract was confirmed, which has important implications for future research directions.

CONCLUSION:

The use of curcumin as an adjuvant in cancer treatment to reduce drug side effects has been studied. The results of the cytotoxic, clonogenic, and migration assays revealed a synergistic effect of Sorafenib and C. aromatica extract. The synergism suggested that the C. aromatica extract could be used to help treat liver cancer.

COMPETING INTEREST:

The authors declare that they have no competing interests

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Received on 28.04.2022 Modified on 18.07.2022

Research J. Pharm. and Tech 2023; 16(1):245-249.

DOI: 10.52711/0974-360X.2023.00045