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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 2  |  Issue : 1  |  Page : 1-5

Specificity and potency of curcumin derivative 64PH in inhibiting HepG2 human hepatocellular carcinoma cell proliferation


1 Department of Gastroenterology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China
2 Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China
3 Department of Industrial Pharmacy, School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, China

Date of Submission15-Dec-2016
Date of Acceptance09-Jan-2017
Date of Web Publication21-Mar-2017

Correspondence Address:
Junwang Xu
Department of Gastroenterology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710049, Shaanxi
China
Weiyi Feng
Department of Pharmacy, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710049, Shaanxi
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ts.ts_38_16

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  Abstract 


Aim: This study aimed to investigate the specificity and potency of curcumin derivative 64PH in inhibiting the proliferation of HepG2 human hepatoma cells in vitro. Methods: Various concentrations of 64PH were administrated to HepG2 hepatoma cells and HL7702 hepatic cells. The viability of cells was evaluated by methyl thiazolyl tetrazolium assay. The concentration-inhibition rates in the two cell lines were calculated, and the accumulation normal distribution function was adopted to fit their rate curves. The differences of the rates between the two cells were observed on the 3rd day of 64PH treatment. The maximum difference and the 95% credibility interval of the corresponding 64PH concentration were evaluated. Results: 64PH inhibited the proliferation of HepG2 and HL7702 cells in vitro. To fit the concentration-effect curves on the 3rd day, the determination coefficients (γ2) were more than 0.99, the half maximal inhibitory concentration (IC50) was 3.07 and 4.28 μg/mL of 64PH, respectively, and their ratio was 1.39. To fit the normal distribution function of the differences of concentration-inhibition rates between HepG2 and HL7702 cells (s2 = 0.9861), the maximum difference of inhibition rates was 33.58%, and the corresponding concentration of 64PH and the 95% credibility interval were 2.65 and 3.52 μg/mL, respectively. Conclusion: In vitro, HepG2 cells are more sensitive than HL7702 cells due to the presence of 64PH. The inhibition of cell proliferation induced by 64PH is stronger in HepG2 than in HL7702 at concentrations between 2.65 and 3.52 μg/mL. 64PH has the potential to be a therapeutic approach in hepatocellular carcinoma and to achieve the desired efficacy and safety.

Keywords: 64PH, concentration, HepG2, HL7702, inhibition rate


How to cite this article:
Li X, Zheng Q, Zhuo Y, Chen J, Ma W, Zhao X, Zhao P, Liu X, Lei H, Xu J, Feng W. Specificity and potency of curcumin derivative 64PH in inhibiting HepG2 human hepatocellular carcinoma cell proliferation. Transl Surg 2017;2:1-5

How to cite this URL:
Li X, Zheng Q, Zhuo Y, Chen J, Ma W, Zhao X, Zhao P, Liu X, Lei H, Xu J, Feng W. Specificity and potency of curcumin derivative 64PH in inhibiting HepG2 human hepatocellular carcinoma cell proliferation. Transl Surg [serial online] 2017 [cited 2017 Oct 17];2:1-5. Available from: http://www.translsurg.com/text.asp?2017/2/1/1/202648




  Introduction Top


Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of death in the world.[1] Because of its complicated molecular pathogenesis, there are limited options for effective systemic treatment of the disease. Curcumin is a phenolic compound from the plant Curcuma longa which is frequently used as a flavoring agent in food.[2] Recently, many publications reported that curcumin can also be a chemopreventive agent and an inhibitor of metastasis in a variety of cancers (hematological, breast, gastrointestinal, liver, and prostate).[3] Emerging evidence has shown that curcumin is a promising agent in the treatment of HCC based on its antioxidant, apoptotic, and anti-inflammatory effects.[4]

Curcumin with its polyphenolic structure is water insoluble and scarcely dissolves in the organic phase. Previous studies addressing the absorption and metabolism of curcumin after oral administration have demonstrated its poor bioavailability in vivo.[5] To solve this problem, we synthesized a series of curcumin derivatives and screened their activities in inhibiting the proliferation of tumor cells in vitro. The results indicated that one of the curcumin derivatives, named 64PH here, which was synthesized from curcumin, tetramethylpyrazine, and N-bromosuccinimide through free radical reaction [Figure 1],[6] presented valid antitumor activity in a range of tumor cell lines. Our further research confirmed that 64PH not only inhibited the proliferation of HCT-8, Bel-7402, BGC-823, A-549, and A2780 tumor cells, but also dramatically suppressed angiogenesis in the chick chorioallantoic membrane model.[6] Because the clinical applications of most chemotherapeutic antitumor agents are often restricted by serious adverse drug reactions due to unspecific anticancer activity,[7] it is crucial to investigate the effects of antitumor agents targeting specific cells. The present study aimed to evaluate the concentration response and specificity of 64PH on inhibiting the proliferation of HepG2 human liver carcinoma cell lines in vitro.
Figure 1: Molecular structural formula of 64PH

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  Methods Top


Reagents

64PH,[6] obtained from the School of Chinese Pharmacy, Beijing University of Chinese Medicine, was dissolved at a concentration of 10 mg/mL in dimethyl sulphoxide (DMSO) at 60°C and was stored at −20°C, protected from light. It was further diluted in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Gaithersburg, USA) to the required concentration immediately before use. Fetal calf serum was provided by Hangzhou Sijiqing Company (Zhejiang, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cell culture

HepG2 and HL-7702 human hepatic immortalized cells (provided by the Cancer Institute Laboratory of the Medical College, Xi'an Jiaotong University, China) were cultured in RPMI-1640 medium with 10% fetal calf serum at 37°C and 5% CO2.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

Cell viability was determined with the MTT assay. Cells were seeded in 96-well plates at a density of 5 × 103 each well and cultured in 200 µL RPMI-1640 medium with 10% fetal calf serum for 24 h at 37°C with 5% CO2. After treatment with different concentration gradient of 64PH (at 1.56, 3.13, 6.25, 12.50, 25.00, and 50.00 µg/mL) for 24, 48, 72, 96, 120 h, respectively the cells were washed twice with phosphate-buffered saline. Then, 180 µL RPMI-1640 and 20 µL MTT (0.5 mg/mL) were added to each well and incubated at 37°C for 4 h. To dissolve formazan crystals, the culture medium was replaced with 150 µL DMSO. After the mixture was shaken at room temperature for 10 min, the optical density (OD) value of absorbance in each well was determined at 490 nm using a microplate reader (BioTek Instruments, EL×808, USA). The data were shown as the average of three independent experiments. The growth inhibition rate is calculated as follows:

Growth inhibition rate IR (%) = (OD value of control group − OD value of experimental group)/OD value of control group × 100%.

Mathematical fitting of the growth inhibition curves

The formula for cumulative normal distribution function of concentration-inhibition rate is as follows:[8]



Where, X is the explanatory variable which represents the logarithm of the drug concentration; its associated dependent variable y is modeled as a random variable with a mean given by F (X). A and D denote the maximum and minimum inhibition rates (%), respectively. The parameter μ in this definition is the mean or expectation of the distribution; σ is its standard deviation, and its variance is therefore σ2. Residual sum of squares (RSS) is the sum of squares (SS) of deviations of predicted values from actual empirical values of data. Regression SS is the SS of deviation from mean minus RSS. Coefficient of determination (γ2) is the regression SS divided by SS.





Based on the least square method, when γ2 reaches its maximum, meaning the overall solution minimizes the SS of errors made in the results of every single equation, the accumulative normal distribution functions were fitted, and the concentration-response curves of HepG2 and HL7702 cells were created using GraphPad Prism 6.0 (Graphpad software, La Jolla, CA, USA).

Comparison of half maximal inhibitory concentration and concentration- response curves between HepG2 and HL7702

Half maximal inhibitory concentration (IC50) represents the concentration of a drug required for 50% inhibition in vitro, which, therefore, is a measure of effectiveness of a compound in inhibiting biological or biochemical function. The maximal efficacy (A value), IC50, and distribution variance (σ2) of 64PH were observed visually by comparing the concentration-effect curves of 64PH on HepG2 and HL7702 cells. The ratio of IC50 values reflected the specificity or safety of 64PH.

Mathematical fitting of inhibition rate difference curves

The parameters A, D, μ, σ, and 95% credibility interval were evaluated based on the least square method when γ2 reached its maximum, and the curves were drawn with GraphPad Prism 6.0 (Graphpad software, La Jolla, CA, USA).

Statistical methods

Data are expressed as the mean ± standard error of the mean. Statistical analyses among groups were performed using one-way analysis of variance with Fisher's least-significant-difference test, and P < 0.05 was considered significant difference.


  Results Top


Inhibitory effects of 64PH on HepG2 and HL7702

Compared with the negative control groups, 64PH inhibited the proliferation of HepG2 and HL7702 cells in vitro, with a statistically significant difference (P < 0.01). As shown in [Figure 2]a, 64PH at the concentrations of 6.25, 12.50, 25.00, and 50.00 µg/mL significantly decreased the growth of HepG2 cells in a time-dependent manner after 48 h (P < 0.01). The inhibition ratio at 48 h reached 68.95%, 76.14%, 87.68%, and 86.07%, respectively. These results indicated that 64PH treatment remarkably inhibited HepG2 cell proliferation. Comparatively, the inhibitory effects of 64PH on HL7702 cells took a longer time. The inhibition ratio at 6.25 µg/mL reached 70.93% after 96 h and that at 50 µg/mL was 85.77% after 72 h [Figure 2]b. Thus, the inhibitory effect of 64PH on HL7702 cells was obviously weaker than that on HepG2 cells. The growth inhibition curves of 64PH on HepG2 cells at concentrations above 6.25 µg/mL are very similar. The inhibition ratio reached 94.84% at 96 h when the HepG2 cells reached saturation.
Figure 2: The inhibition rates of 64PH on HepG2 cells (a) and HL7702 cells (b)

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Mathematical fitting of the growth-inhibition curves

The cells reached saturation on day 4 and entered the period of stagnation. Therefore, we fitted the differences of concentration-response rates between HepG2 and HL7702 with the normal distribution on day 3, i.e., at 72 h. The best-fit curves are shown in [Figure 3]. The best-fit values of A, D, μ, σ, and γ2 in the 64PH-on-HepG2 curve were 87.01%, −1.94%, 0.5644, 0.0621, and 1.000, respectively, and those in the 64PH-on-HL7702 curve were 87.41%, −17.20%, 0.7509, 0.0649, and 0.9984, respectively.
Figure 3: The fitting curves of the concentration-inhibition rates of 64PH on HepG2 and HL7702 cells and their differences

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Comparison of the parameters of concentration-response curves between HepG2 and HL7702 cells

The IC50 of 64PH on HepG2 was 3.07 µg/mL and that on HL7702 was 4.28 µg/mL. From the concentration-effect curves, a ratio of 1–1.39 was present in the IC50 on HepG2 and HL7702 cells. These data indicated a stronger inhibitory effect of 64PH on HepG2 cells than on HL7702 cells. This might suggest that 64PH targeting liver carcinoma has a strong effect on tumor cells but minimal adverse effects on normal liver cells.

Fitting the normal distribution function of the difference of concentration-inhibition rate curves

The differences of concentration-inhibition rate curves between HepG2 and HL7702 cells were calculated. The values of μ, σ2, and γ2 were 0.0193, 0.4847, and 0.9861, respectively. When the difference in inhibition rate reached a maximum of 33.58%, the concentration corresponding to the parameter μ was 3.05 µg/mL. The 95% credibility interval was 0.42–0.55, and the corresponding concentration of 64PH was 2.65–3.52 µg/mL.


  Discussion Top


Drugs targeting cancer-specific pathways need to have not only strong antitumor effects but also minimal adverse effects against normal cells so as to achieve the desired efficacy and safety.[9] The safety concern warrants careful evaluation of the specificity and intensity of the drugs. In our previous research, a curcumin derivative, 64PH, showed significant antitumor effect on HepG2 hepatoma cells in vitro and on H22 cells in vivo.[10] However, it was not clear how this compound would function on normal hepatocytes. To get the answer and determine whether 64PH could be a therapeutic option for hepatoma, we conducted the present study, in which HepG2 hepatoma cells and normal HL7702 hepatic cells were employed to compare the effects of 64PH on tumor cells versus normal cells. The effective concentration ranges and cytotoxic specificity of 64PH on both cell lines were evaluated. The results showed that IC50 of HepG2 and HL7702 cells was 3.07 and 4.28 µg/mL, respectively. The differences of concentration-inhibition rates between the two cell lines were fitted with the normal distribution on day 3 because cells reached saturation on day 4. All the determination coefficients (γ2) were more than 0.98. Within the range of concentration from 2.65 to 3.52 µg/mL, the cell proliferation inhibition induced by 64PH was significantly stronger in HepG2 than in HL7702, indicating that 64PH was more cytotoxic to the tumor cells than to the normal cells at the given concentration range. These results are in consistent with those seen in a previous study where HepG2 cells decreased by 84% when exposed to 100 mg/L curcumin as compared with being exposed to normal medium, while HL7702 cells were less sensitive to the curcumin treatment.[11] In addition, the study also reported that 64PH showed the activity in selectively killing tumor cells rather than normal cells, which suggested that 64PH might be a potential drug in hepatoma therapy.

Curcumin is a free radical scavenger and hydrogen donor and exhibits both pro- and anti-oxidant activity.[12] It also binds metals, particularly iron and copper, and can function as an iron chelator.[12] Although the details of mechanisms underlying the inhibition of tumorigenesis by curcumin remain unclear, it has been proven that a combination of anti-oxidant, anti-proliferative, pro-apoptotic, and anti-angiogenetic properties is involved in the regulation of genes and molecules associated with multiple signaling pathways [13] such as those regulated by NF-kappaB, Akt, growth factors, and cytoprotective pathways dependent on Nrf2.[14] In addition, curcumin also has inhibitory effects on cell cycle progression, predominantly at the level of the G2/M transition point.[15] It was reported that curcumin monotherapy failed to stimulate the regenerative process after partial hepatectomy with cecal ligation and puncture, and it even suppressed hepatocellular proliferation after partial hepatectomy with otherwise undisturbed regeneration.[15] The suppression of liver regeneration, especially of the mitotic activity after liver resection by perioperative curcumin treatment, has also been reported by other researchers.[16] Furthermore, curcumin exhibits the anti-proliferative property by regulating multiple cellular functional pathways involving proliferation, survival, and various protein pathways, among others.[17] The discrepant effects of curcumin derivative 64PH on HepG2 and HL7702 cells, as revealed in this study, may be based on different threshold values in the induction of anti-proliferative, pro-apoptotic, and anti-oxidant factors, among others.

Preclinical animal experiments and phase I clinical trials have demonstrated that curcumin does not show any toxicity to humans when taken orally at a dose up to 8000 mg/day for 3 months.[18] The cytotoxicity of curcumin to HL7702 cells was much lower than that of cisplatin.[19] Our previous data have shown that the novel compound 64PH exerts weaker toxicity toward HL7702 cells than does curcumin. However, its range of safe and effective concentration is too narrow. As a result, it is necessary to develop new generations of the antitumor drugs from the molecular modification of curcumin. Amphiphilic 64PH drug nanoparticle carriers maybe also healthier and more effective to decrease HCC.[20] However, this present study provided a new research method which can at least assist in evaluating the safety and effective concentration range of other antitumor drugs.

Taken together, the above findings disclose that HepG2 cells are more sensitive in terms of cytotoxicity to 64PH, while HL7702 cells exhibit a higher healthy viability after 64PH treatment in vitro. The inhibition of cell proliferation by 64PH in HepG2 was stronger than in HL7702 at the concentrations between 2.65 µg/mL and 3.52 µg/mL. The present study confirms the potential of using the curcumin derivative 64PH as a therapeutic option for hepatic carcinoma and provides basis for further exploration of 64PH in vivo.

Acknowledgment

The authors thank Penglong Wang and Kuo Xu for their initial work on the synthesis of 64PH and Zongchang He and Qi Chen for the language-editing organizations.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Qian H, Yang Y, Wang X. Curcumin enhanced adriamycin-induced human liver-derived hepatoma G2 cell death through activation of mitochondria-mediated apoptosis and autophagy. Eur J Pharm Sci 2011;43 (3):125-31.  Back to cited text no. 1
    
2.
Ning L, Wentworth L, Chen H, Weber SM. Down-regulation of Notch1 signaling inhibits tumor growth in human hepatocellular carcinoma. Am J Transl Res 2009;1 (4):358-66.  Back to cited text no. 2
    
3.
Charpentier MS, Whipple RA, Vitolo MI, Boggs AE, Slovic J, Thompson KN, Bhandary L, Martin SS. Curcumin targets breast cancer stem-like cells with microtentacles that persist in mammospheres and promote reattachment. Cancer Res 2014;74 (4):1250-60.  Back to cited text no. 3
    
4.
Hu Y, Wang S, Wu X, Zhang J, Chen R, Chen M, Wang Y. Chinese herbal medicine-derived compounds for cancer therapy: A focus on hepatocellular carcinoma. J Ethnopharmacol 2013;149 (3):601-12.  Back to cited text no. 4
    
5.
Lin CC, Lin HY, Chen HC, Yu MW, Lee MH. Stability and characterisation of phospholipid-based curcumin-encapsulated microemulsions. Food Chem 2009;116 (4):923-8.  Back to cited text no. 5
    
6.
Wang P, She G, Yang Y, Li Q, Zhang H, Liu J, Cao Y, Xu X, Lei H. Synthesis and biological evaluation of new ligustrazine derivatives as anti-tumor agents. Molecules 2012;17 (5):4972-85.  Back to cited text no. 6
    
7.
Liu J, Xu H, Zhang Y, Chu L, Liu Q, Song N, Yang C. Novel tumor-targeting, self-assembling peptide nanofiber as a carrier for effective curcumin delivery. Int J Nanomed 2014;9:197-207.  Back to cited text no. 7
    
8.
Fang C, Liu L, Ding H. The Concentration-effect and specificity of arsenic trioxide inhibited the proliferation of human malignant fibroblasts in vitro. China Pharm 2013;22 (13):12-4.  Back to cited text no. 8
    
9.
Jin F, Gao D, Zhang C, Liu F, Chu B, Chen Y, Chen YZ, Tan C, Jiang Y. Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl) phenyl)-3-phenylurea derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor treatment. Bioorg Med Chem 2013;21 (3):824-31.  Back to cited text no. 9
    
10.
Li XQ, Lei HM, Wang PL, Xu K, Zhuo YC, Chen JG, Zheng QW, Ma WB, Zhao X, Xu JW, Feng WY. Anti-tumor effects of curcumin derivative 64PH. Chin Hosp Pharm J 2015;35 (17):8-12.  Back to cited text no. 10
    
11.
Guo ZS, Shao HZ, Xue F, Lu CX. Jianghuangsu exerts anti-proliferative and pro-apoptotic effects on HepG2 cells through extrinsic and intrinsic pathways. Chin J Exp Surg 2012;29 (5):851-3.  Back to cited text no. 11
    
12.
Hatcher H, Planalp R, Cho J, Torti FM, Torti SV. Curcumin: From ancient medicine to current clinical trials. Cell Mol Life Sci 2008;65 (11):1631-52.  Back to cited text no. 12
    
13.
Sarkar FH, Li Y, Wang Z, Padhye S. Lesson learned from nature for the development of novel anti-cancer agents: Implication of isoflavone, curcumin, and their synthetic analogs. Curr Pharm Des 2010;16 (16):1801-12.  Back to cited text no. 13
    
14.
Bar-Sela G, Epelbaum R, Schaffer M. Curcumin as an anti-cancer agent: Review of the gap between basic and clinical applications. Curr Med Chem 2010;17 (3):190-7.  Back to cited text no. 14
    
15.
Seehofer D, Neumann UP, Schirmeier A, Carter J, Cho SY, Lederer A, Rayes N, Menger MD, Nüssler AK, Neuhaus P. Synergistic effect of erythropoietin but not G-CSF in combination with curcumin on impaired liver regeneration in rats. Langenbecks Arch Surg 2008;393 (3):325-32.  Back to cited text no. 15
    
16.
Seehofer D, Schirmeier A, Bengmark S, Carter J, Koch M, Glanemann M, Nüssler AK, Neuhaus P, Menger MD. Inhibitory effect of curcumin on early liver regeneration following partial hepatectomy in rats. J Surg Res 2009;155 (2):195-200.  Back to cited text no. 16
    
17.
Hung CT, Huang SM, Cheng HC, Liu ST, Chang YL, Liu YC, Wang WM. The inhibitory mechanism by curcumin on the Zac1-enhanced cyclin D1 expression in human keratinocytes. J Dermatol Sci 2015;79 (3):262-7.  Back to cited text no. 17
    
18.
Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, Ko JY, Lin JT, Lin BR, Ming-Shiang W, Yu HS, Jee SH, Chen GS, Chen TM, Chen CA, Lai MK, Pu YS, Pan MH, Wang YJ, Tsai CC, Hsieh CY. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res 2001;21 (4B):2895-900.  Back to cited text no. 18
    
19.
Zhou T, Ye L, Bai Y, Sun A, Cox B, Liu D, Li Y, Liotta D, Snyder JP, Fu H, Huang B. Autophagy and apoptosis in hepatocellular carcinoma induced by EF25-(GSH) 2: A novel curcumin analog. PLoS One 2014;9 (9):e107876.  Back to cited text no. 19
    
20.
Chang R, Sun L, Webster TJ. Short communication: Selective cytotoxicity of curcumin on osteosarcoma cells compared to healthy osteoblasts. Int J Nanomed 2014;9:461-5.  Back to cited text no. 20
    


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  [Figure 1], [Figure 2], [Figure 3]



 

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