Anti HCV activity and expression inhibition of HCC markers by protein extract from Iberis gibraltarica

Atividade anti-HCV e inibição da expressão de marcadores HCC por extrato de proteína de Iberis gibraltarica

Abstract

Hepatitis C virus infection (HCV) is the foremost reason of progressive hepatic fibrosis and cirrhosis, with an elevated risk of hepatocellular carcinoma (HCC) development. Medicinal plants have been used for human health benefits for several years, but their therapeutic potential needs to be explored. The main objective of this study was to figure out the in vitro antiviral and anticancer characteristics of total crude protein of Iberis gibraltarica against HCV and HCC. Total crude protein of Iberis gibraltarica was isolated and quantified. The level of cytotoxicity was measured against the HepG2 cell line and it shows no significant cytotoxicity at the concentration of 504µg/ml. The anti-HCV effect was determined by absolute quantification via real time RT-PCR method and viral titer was reduced up to 66% in a dose dependent manner against the total protein of Iberis gibraltarica. The anticancer potential of Iberis gibraltarica was also examined through mRNA expression studies of AFP and GPC3 genes against the total protein of Iberis gibraltarica-treated HepG2 cells. The results show up to 90% of the down-regulation expression of AFP and GPC3. The obtained results indicate the therapeutic potential of total protein of Iberis gibraltarica against HCV and hepatocellular carcinoma in vitro.

Keywords:
HCV; HCC; Iberis gibraltarica; HepG2 and Real time PCR

Resumo

A infecção pelo vírus da hepatite C (HCV) é a principal causa de fibrose hepática progressiva e cirrose, com risco elevado de desenvolvimento de carcinoma hepatocelular (HCC). As plantas medicinais vêm sendo utilizadas para benefícios à saúde humana há vários anos, mas seu potencial terapêutico precisa ser explorado. O principal objetivo deste estudo foi descobrir as características antivirais e anticancerígenas in vitro da proteína bruta total de Iberis gibraltarica contra HCV e HCC. A proteína bruta total de Iberis gibraltarica foi isolada e quantificada. O nível de citotoxicidade foi medido contra a linha celular HepG₂ e não apresenta citotoxicidade significativa na concentração de 504µg/ml. O efeito anti-HCV foi determinado por quantificação absoluta através do método RT-PCR em tempo real e o título viral foi reduzido em até 66% de forma dose-dependente contra a proteína total de Iberis gibraltarica. O potencial anticancerígeno de Iberis gibraltarica também foi examinado através de estudos de expressão de mRNA dos genes AFP e GPC₃ contra a proteína total de células HepG₂ tratadas com Iberis gibraltarica. Os resultados mostram até 90% da expressão de regulação negativa de AFP e GPC₃. Os resultados obtidos indicam o potencial terapêutico da proteína total de Iberis gibraltarica contra HCV e carcinoma hepatocelular in vitro.

Palavras-chave:
HCV; HCC; Iberis gibraltarica; HepG₂ e PCR em tempo real

1. Introduction

One of the foremost bases of the chronic liver diseases is the hepatitis C virus (HCV), a hepatotrophic RNA virus. HCV is a small, enveloped, single-stranded, positive-sense RNA virus and a associate of the family Flaviviridae with a genome size of 9.6 kb. Among the different subtypes of HCV, genotype 3a is more prevalent in Pakistan (Alecu et al., 1998). About 170 million people around the world are believed to have been infected with HCV (Umer and Iqbal, 2016). HCV causes various diseases of the liver, which account for half a million deaths annually (Curry et al., 2015). There is currently no HCV vaccine available and combination therapy, such as pegylated interferon and ribavirin, has previously been used for HCV treatment (Manns et al., 2006), but not for all HCV genotypes (Palumbo, 2011). Direct-acting antivirals (DAAs) have been introduced for the management of chronic HCV. Boceprivir and telaprevir have significantly improved HCV treatment by inhibiting HCV NS3/4A proteases, although these drugs have shown serious side effects (Shaikh and Shih, 2012) . More recently, sofosbuvir and ledipasvir have been approved for HCV treatment with improved results and have shown 96-99% sustained virologic response in 12 to 24 weeks (Scott, 2018). But the high cost of HCV care leaves a large number of HCV-infected people out of control, most of whom are in resource-poor countries such as South East Asia and Africa with very high burdens on HCV-positive people (WHO, 2017). Interferon and DAAs have many severe side effects (Sa-Ngiamsuntorn et al., 2016) however, viral resistance strains called Resistance Associated Strains emerge due to the heterogeneous pool of HCV, which explain their resistance to DAA treatment (Loggi et al., 2018).

The major complication of HCV virus infection is hepatocellular carcinoma (HCC) with significant rates of mortality and morbidity (Chen and Morgan, 2006). The prevalence rate of HCC is very high and accounts for 75% of cases of hepatic cancer (Jemal et al., 2011; Maillard, 2011). In addition, more than 80% of this cancer has also surfaced in developing economies where HCC prognosis is poor, with some other factors (Rehman et al., 2011) such as metabolic disorders such as metabolic syndrome, non-alcoholic fatty liver disease, obesity and diabetes increasing. (Yang and Roberts, 2010). Systematic treatment solutions for HCC included liver transplantation, chemotherapy, radiation and percutaneous medications, but these treatments remain largely inadequate. A variety of chemotherapy drugs against HCC have been reported (Peters et al., 2002). Besides, these drugs have also been associated with their side effects that are harmful to normal cells. Medicinal plants have been shown to reduce the threat of HCC and liver cirrhosis (Umer and Iqbal, 2016) . Most of the research has been focused on developing more effective anti-viral and anticancer agents with selective toxicity (Senthilraja and Kathiresan, 2015, Vijayan et al., 2004). The number of plants with anticancer activity has been reported, but few of them include Cannabis sativa (Galve-Roperh et al., 2000), Bolbostemma paniculatum (Cheng et al., 2006), Apis mellifera (Hamzaoglu et al., 2000), Astralagus hedysarum (Wang et al., 1989), Hypericum perforatum (Alecu et al., 1998) and Camellia sinensis (Esghaei et al., 2018) . Iberis gibraltarica relates to the genus Iberis, a member of the Brassicaceae family (Saeed et al., 2021). Iberis amara extracts have been reported to show anticancer activity (Weidner et al., 2016). The main objective of the current study was to examine the anticancer and antiviral potential of Iberis gibraltarica crude protein extract.

2. Materials and Methods

2.1. Total protein isolation

Iberis gibraltarica seeds were brought by the local seller (MOREGREEN, 2017) and cultivated in the CAMB field and subsequently verified by Dr. Zahoor Sajid (University of the Punjab). Three different buffers (Kim et al., 2011) such as Buffer 1; 100mM citrate buffer (pH3.0) containing 50mM NaCl, Buffer 2; 100mM acetate buffer (pH5.0) containing 50mM NaCl and Buffer 3 was comprised of 100mM phosphate buffer (pH7.0) containing 50mM NaCl were used to isolate the total crude protein. Seeds were ground to a fine powder with a pestle and mortar and mixed with each buffer in a proportion of 1:10 (w/v) for 2 hours at 4°C. Muslin cloth was used to remove the seed debris, and the supernatant was collected by centrifugation (Eppendorf 5415R, Germany) at 4°C for 30 minutes at 13000 rpm.

Bradford method was performed to find out the total protein concentration using Bradford Reagent (Bio-Rad, USA) (Bradford, 1976). Collected supernatant of individual pH was dialyzed separately overnight by dialyzing tube (Spectra/Por RC Biotech membrane, 6-8 kDa MWCO, USA) against 10mM of citrate buffer pH3.0, phosphate buffer of pH 7.0 and acetate buffer pH 5.0 at 4°C. Total protein isolated from distinct buffers was analyzed by SDS-PAGE (Smith, 1984).

2.2. Cell culture

The HepG2 cells were cultured using the Dulbecco’s modified Eagle medium (DMEM) containing 100U/ml penicillin, 100 µg/ml streptomycin and 10% Fetal Bovine Serum (FBS) at 37°C in 5% CO2 incubator at 37˚C in the cell culture lab, CAMB.

2.3. Cytotoxicity assay

The level of cytotoxicity of the protein extract was measured by the MTT (3-(4,5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide) assay kit (Millipore, USA). DMEM was used to culture 100µl (1×10⁵) of HepG2 cells in 96 wells flat bottom plate (Corning, USA). Cells were grown at 37°C for 24 hours in a CO₂ incubator (BINDER, Germany). Different concentrations of the protein were supplemented to the 96-wells plate and then placed in a CO₂ incubator for 24 hours at 37 °C. For each dilution, three replicates were analyzed. The media was carefully removed from the wells and changed to 100μl of newly prepared media after 24 hours, as directed by the manufacturer, 10µL MTT solution (5mg/ml in PBS) was supplemented and incubated again in a CO₂ incubator at 37°C for 4 hours. Subsequently, wells were filled with 0.1ml of DMSO (Dimethyl sulfoxide) to dissolve the formazen crystals. The optical density of the MTT formazan product was taken by an ELISA (Enzyme-linked immunosorbent assay) reader (SpectraMax Plus 96, Molecular Devices, USA) with a reference wavelength of 620 nm and a test wavelength of 570 nm (Rehman et al., 2011).

2.4. Anti-HCV activity

HepG2 cells (3×10⁵ cells/well) were grown in the 60mm cell culture dish. After attaining the 60-70% confluency, washed the cells with 1×PBS and infected with 500µl HCV (3a genotype) infected sera (1×10⁵ IU/well) along with 500µl serum free media for 24 hours (El-Awady et al., 2006; Noreen et al., 2015). On the following day, wash the adherent cells with PBS twice and left the cells to grow for 48 hours.

In order to analyze the effects of total protein of I.gibraltarica, the infected HepG2 cells were left to grow in a 6-well plate. At the 60-70% confluency, treated the cells with different concentrations of total protein of I.gibraltarica (30, 60 and 90µg/ml) along with the control (without protein) for 24 hours. The total RNA was then isolated from each well using the Qiagen RNA isolation kit as instructed by the manufacturer. Viral quantification was performed using the Qiagen HCV quantitative analysis kit. For viral RNA quantification, 10µl of the total RNA extracted from cell lysate was mixed with inhibitory concentration (IC) (described above) and quantified with Rotor-Gene Q 2plex HRM System (Qiagen, USA).

2.5. Relative quantification of AFP and GPC3 gene expression

HepG2 cells were seeded at a density of 1×10⁵ in a 12-well plate and incubated at 37°C for 24 hours. Subsequently, the media was replaced and inoculated for another 24 hours with different concentrations (30, 60 and 90μg / ml) of crude protein extracts along with a protein-free buffer control in the CO2 incubator. Total RNA was extracted from protein-treated cells using the TRIzol Reagent (Invitrogen, USA) (Rio et al., 2010). Quantification of the extracted RNA was performed with the Nano drop (ND-1000, OptiPlex, USA) followed by synthesis of cDNA through the Revert Aid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Lithuania) as instructed by the manufacturer.

2.6. Real time PCR analysis

The Alpha-fetoprotein (NM_001134) and Glypican 3 (NM_004484) mRNA expressions were evaluated in the protein-treated cells using real time PCR (PikoReal™ Thermo Fisher, Finland). β- Actin (NM_001101) was used as a reference gene (Li et al., 2010). Gene expression was analyzed by real-time PCR analysis with Maxima SYBR Green qPCR Master Mix (Thermo Scientific). For AFP RT-PCR amplification, one cycle of 95°C for 5 minutes was followed by 35 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Similarly, for GPC3 RT-PCR amplification, one cycle of 95°C for 5 minutes was followed by 35 cycles of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds. The relative gene expression levels were determined using the 2^–∆∆Ct method. Table 1 (Supplementary material) lists the primers that were applied.

Table 1
List of primers used for the real time PCR study.

Primers	Sequences	Amplicon size
AFP Forward	5/-TGTCCCTCCTGCATTCTCTG-3/	189
AFP Reverse	5/-TGGCAGCATTTCTCCAACAG-3/
GPC3 Forward	5/-TACTGCTCTTACTGCCAGGG-3/	186
GPC3 Reverse	5/-ACCAAGCAGTACGTTCTCCA-3/
β-Actin Forward	5/-CCCTGGAGAAGAGCTACGAG-3/	180
β-Actin Reverse	5/-CGTACAGGTCTTTGCGGATG-3/

2.7. Statistical analysis

GraphPad Prism 7 software was used for statistical analyses. Cytotoxicity and anti-HCV experiments were carried out in triplicate, while AFP and GPC3 relative expression studies were performed in duplicate. The mean and standard deviations of the data were determined using descriptive statistics. One way ANOVA was used to explore the significant reduction in mRNA expression of the AFP and GPC3 genes at P≤0.05.

3. Results

3.1. SDS-PAGE analysis

In order to evaluate the maximum protein profile, the total protein was isolated by means of three distinct buffers (citrate buffer (pH3.0), acetate buffer (pH5.0) and phosphate buffer (pH7.0). Figure 1 (Supplementary material) shows the results of the SDS-PAGE for Iberis gibraltarica total protein. The total crude protein profile obtained by SDS-PAGE at three different pH levels is shown in Figure 1A. Figure 1B (Supplementary material) illustrates the silver staining results from three different pH levels. SDS-PAGE results from crude extracts revealed that maximum protein extraction was achieved at pH7.0, as indicated by the maximum number of protein bands and silver staining results. The pH7.0 buffer was therefore used to further isolate the protein from I.gibraltarica.

Figure 1
(A) indicates the SDS-PAGE analysis of I.gibraltarica total protein using three different buffers. Lane 1 represents the protein marker (Cat. No. 26616, Thermo, Lithuania). Lanes 2, 3 and 4 represent total protein isolated by citrate buffer (pH3.0), acetate buffer (pH5.0) and phosphate buffer (pH7.0) respectively; (B) represents the silver staining.

3.2. Cytotoxicity assay

The cytotoxic effect of Iberis gibraltarica total protein is shown in the Figure 2. The most considerable effect was achieved by up to 84% cell viability after 24 hours, with a maximum concentration of I. gibraltarica total protein of 504 μg/ml. A higher protein concentration can also be used for a more pronounced effect. Hence, no significant decrease in cell viability with respect to control was observed after 24 hours.

Figure 2
Cytotoxic effect of total protein of I.gibraltarica in HepG2 cells.

3.3. Anti -HCV Activity of I.gibraltarica

Total protein of I.gibraltarica was evaluated for antiviral activity against the HCV. The results obtained after real time RT-PCR shows that total protein of I.gibraltarica reduces the virus RNA for 20, 48 and 66% at the concentration of 30, 60 and 90µg/ml respectively, as shown in the Figure 3.

Figure 3
Antiviral effect of total protein of I.gibraltarica against HCV in HepG2 cells (liver cells). Real time PCR profile generated, and bars in the graph depict the standard deviation while, asterisks (**) indicates the P<0.01 and asterisks (****) indicate a significant decrease at P<0.0001 in HCV titer at 30, 60 and 90µg/ml doses of the total protein of I.gibraltarica.

3.4. AFP and GPC3 gene expression

A major decrease in GPC3 and AFP gene expression was observed in cells treated with I.gibraltarica complete protein. Delta delta method was used for qPCR analysis. Decreased AFP expression was up to 92%, while GPC3 expression was observed in a dose-dependent manner as shown in Figure 4.

Figure 4
Effect of total protein of I.gibraltarica on AFP and GPC3 gene expression. Real time PCR profile generated, and bars in the graph depict the standard deviation while, **** indicate a significant decrease at P<0.0001 in AFP and GPC3 gene expressions at 30, 60 and 90µg/ml doses of the total protein of I.gibraltarica.

Melting curve analysis showing the melting temperature peaks (Tm) of AFP and GPC3 genes in Figure 1S and 2S, respectively.

One way ANOVA was applied, and it shows that all the results obtained are statistically significant at P<0.0001.

4. Discussion

Plant-based substances such as small molecular compounds and proteins, have been proposed as effective medicines for cancer management in response to many of the adverse effects of conventional cancer treatments, such as chemotherapy and radiation therapy (Hernandez-Ledesma et al., 2013). A number of plants proteins are recognized for their anticancer and antiviral nature, a plant lectin purified from Praecitrullus fistulosus has been observed to inhibit tumor growth in vivo (Shivamadhu et al., 2017) and an antiviral protein (RIP type I) isolated from Phytolacca americana significantly inhibited the levels of TMV in Nicotiana benthamiana plant leaves. However, there are numerous plants that should be evaluated for their activities as Pokeweed, because Pokeweed contains some toxic components, and its therapeutic potential should be carefully evaluated (Zhu et al., 2016).

The maximum protein profile and concentration obtained at pH 7 buffer through SDS-PAGE and Bradford assay using Bradford Reagent (BioRad, USA) . Similar studies were carried out with bioactive protein extractions from different plant sources (Zekri et al., 2009) . Previously, using the hydroethanolic extract of I.amara has been examined against the colon cancer (Weidner et al., 2016).

Presently, no information is available on the bioactivity of I.gibraltarica seed protein extract. This study is the first antiviral and anticancer report on the potenial of I.gibraltarica seed proteins. The cytotoxic behavior of total protein isolated from I.gibraltarica was assessed using the HepG2 cells. The least cell viability percentage was measured at a concentration of 525µg/ml after 24 hours. The cytotoxicity was induced in a concentration dependent manner. Similar results have been observed in the protein extract of Morinda pubescens, which shows the maximum cell viability of the Vero cells at a concentration of 100μg/ml (Thomas et al., 2017). Another protein known as Galanthus nivalis agglutinin showed a minimal inhibitory effect on murine and human cancer cell lines when tested for cytotoxicity (Sandström et al., 2004).

Various plant species associated with different taxonomic families are found to generate endogenous, non-stress-induced inhibitor proteins called antiviral proteins (AVPs) (Choudhary et al., 2008). Certain plant proteins have demonstrated antiviral activity against a variety of plant viruses (Bilal et al., 2020; Lusvarghi and Bewley, 2016). Different plant protein such as lectins and ribosome inactivating proteins (RIPs) have also been shown the antiviral activities against the human infected viruses (Alecu et al., 1998; Al-Sohaimy et al., 2007). We evaluated the in vitro anti-HCV potential of the total protein of I. gibraltaricain hepatitis C virus infected HepG2 cells (Tang et al., 2008). The results of the Real Time RT-PCR show the total I. gibraltarica protein reduces the RNA virus level up to 66% at 90μg/ml. Similar results have been reported using the plant proteins against HCV in vitro studies (Al-Sohaimy et al., 2007; Ashfaq et al., 2011). Plant-based chemoprevention and treatment have evolved into useful and effective methods for the treatment and controlling cancer (Pongthanapisith et al., 2013). Plant peptides and proteins are among the most recent natural products with significant anti-carcinogenic activity (Guarneri and Conte, 2004).

The elevated levels of AFP has also been identified as the primary cause of HCC (Henderson et al., 2003) and the ability of its mRNA as a marker in HCC tumor cells has been used for reverse transcription PCR (RT-PCR) (Debruyne and Delanghe, 2008). AFP targeting with siRNA was considered to be a potential therapeutic approach to HCC (Tang et al., 2008). Various studies have shown that GPC3 protein facilitates the spread of HCC cells through the formation of a Wnt complex (Wingless-related integration site) (Ho et al., 2007). (Capurro et al., 2005). In HCC patients, GPC3 translational management offers an effective clinical option (Debruyne and Delanghe, 2008). Furthermore, GPC3 silencing has also been documented to stimulate cell apoptosis (Sun et al., 2011). In this study, relative quantification of AFP and GPC3 genes was carried out in I.gibraltarica crude protein treated HepG2 cells. Real-time PCR findings represent a significant dose-dependent control of AFP and GPC3 expressions (Ruan et al., 2011). Similarly, significant decreases in AFP and GPC3 levels have also been reported in the treatment of Gingko Biloba leaf extract (Wang et al., 1989) . The I.gibraltarica crude protein cytotoxic values indicated the safe limit and worth for further testing (Abushouk et al., 2017). Therefore, purified individual protein/s from total protein of I.gibraltarica may be useful as antiviral and anticancer agents (Liu et al., 2012).

5. Conclusion

The total protein of I. gibraltarica was isolated and evaluated its antiviral and anticancer potential in vitro. Our results show that the total protein of Iberis gibraltarica exhibits significant antiviral activity and down-regulates HCC biomarkers. Our initial findings are very prominent and may open up a new event for further investigation of the individual protein(s) of I. gibraltarica as a natural potent therapeutic candidate for viral and cancer treatment.

Supplementary Material

Supplementary material accompanies this paper.

Figure 1S Melting curve analysis showing the melting temperature peaks (Tm) of AFP gene. Figure 2S Melting curve analysis showing the melting temperature peaks (Tm) of GPC3 gene.

This material is available as part of the online article from https://www.scielo.br/j/bjb

Acknowledgements

We thank HEC, University of the Punjab and CAMB for providing the setup and funding for our work.

References

• ABUSHOUK, A.I., NEGIDA, A., AHMED, H. and ABDEL-DAIM, M.M., 2017. Neuroprotective mechanisms of plant extracts against MPTP induced neurotoxicity: future applications in Parkinson’s disease. Biomedicine and Pharmacotherapy, vol. 85, pp. 635-645. http://dx.doi.org/10.1016/j.biopha.2016.11.074 PMid:27890431.
  » http://dx.doi.org/10.1016/j.biopha.2016.11.074
• ALECU, M., URSACIUC, C., HALALAU, F., COMAN, G., MERLEVEDE, W., WAELKENS, E. and DE WITTE, P., 1998. Photodynamic treatment of basal cell carcinoma and squamous cell carcinoma with hypericin. Anticancer Research, vol. 18, no. 6B, pp. 4651-4654. PMid:9891535.
• AL-SOHAIMY, S., HAFEZ, E., ABDELWAHAB, A. and EL-SAADANI, M., 2007. Anti-HCV lectin from Egyptian Pisum sativum. Australian Journal of Basic and Applied Sciences, vol. 1, pp. 213-219.
• ASHFAQ, U.A., MASOUD, M.S., KHALIQ, S., NAWAZ, Z. and RIAZUDDIN, S., 2011. Inhibition of hepatitis C virus 3a genotype entry through Glanthus Nivalis Agglutinin. Virology Journal, vol. 8, pp. 248. http://dx.doi.org/10.1186/1743-422X-8-248 PMid:21599979.
  » http://dx.doi.org/10.1186/1743-422X-8-248
• BILAL, M., NASIR, I., TABASSUM, B., AKREM, A., AHMAD, A. and ALI, Q., 2020. Cytotoxicity and in-vitro antiviral activity of lectin from crocus Vernus L. against potato virus Y. Applied Ecology and Environmental Research, vol. 18, no. 1, pp. 1301-1315. http://dx.doi.org/10.15666/aeer/1801_13011315
  » http://dx.doi.org/10.15666/aeer/1801_13011315
• BRADFORD, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, vol. 72, no. 1-2, pp. 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3 PMid:942051.
  » http://dx.doi.org/10.1016/0003-2697(76)90527-3
• CAPURRO, M.I., XIANG, Y.-Y., LOBE, C. and FILMUS, J., 2005. Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling. Cancer Research, vol. 65, no. 14, pp. 6245-6254. http://dx.doi.org/10.1158/0008-5472.CAN-04-4244 PMid:16024626.
  » http://dx.doi.org/10.1158/0008-5472.CAN-04-4244
• CHEN, S.L. and MORGAN, T.R., 2006. The natural history of hepatitis C virus (HCV) infection. International Journal of Medical Sciences, vol. 3, no. 2, pp. 47-52. http://dx.doi.org/10.7150/ijms.3.47 PMid:16614742.
  » http://dx.doi.org/10.7150/ijms.3.47
• CHENG, G., ZHANG, Y., ZHANG, X., TANG, H.-F., CAO, W.-D., GAO, D.-K. and WANG, X.-L., 2006. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells. Bioorganic & Medicinal Chemistry Letters, vol. 16, no. 17, pp. 4575-4580. http://dx.doi.org/10.1016/j.bmcl.2006.06.020 PMid:16784856.
  » http://dx.doi.org/10.1016/j.bmcl.2006.06.020
• CHOUDHARY, N., KAPOOR, H.C. and LODHA, M.L., 2008. Cloning and expression of antiviral/ribosome-inactivating protein from Bougainvillea xbuttiana. Journal of Biosciences, vol. 33, no. 1, pp. 91-101. http://dx.doi.org/10.1007/s12038-008-0025-8 PMid:18376074.
  » http://dx.doi.org/10.1007/s12038-008-0025-8
• CURRY, M.P., O’LEARY, J.G., BZOWEJ, N., MUIR, A.J., KORENBLAT, K.M., FENKEL, J.M., REDDY, K.R., LAWITZ, E., FLAMM, S.L., SCHIANO, T., TEPERMAN, L., FONTANA, R., SCHIFF, E., FRIED, M., DOEHLE, B., AN, D., MCNALLY, J., OSINUSI, A., BRAINARD, D.M., MCHUTCHISON, J.G., BROWN JUNIOR, R.S., CHARLTON, M. and ASTRAL-4 Investigators, 2015. Sofosbuvir and velpatasvir for HCV in patients with decompensated cirrhosis. The New England Journal of Medicine, vol. 373, no. 27, pp. 2618-2628. http://dx.doi.org/10.1056/NEJMoa1512614 PMid:26569658.
  » http://dx.doi.org/10.1056/NEJMoa1512614
• DEBRUYNE, E.N. and DELANGHE, J.R., 2008. Diagnosing and monitoring hepatocellular carcinoma with alpha-fetoprotein: new aspects and applications. Clinica Chimica Acta; International Journal of Clinical Chemistry, vol. 395, no. 1-2, pp. 19-26. http://dx.doi.org/10.1016/j.cca.2008.05.010 PMid:18538135.
  » http://dx.doi.org/10.1016/j.cca.2008.05.010
• EL-AWADY, M.K., TABLL, A.A., EL-ABD, Y.S., BAHGAT, M.M., SHOEB, H.A., YOUSSEF, S.S., BADER EL-DIN, N.G., REDWAN, R.M., EL-DEMELLAWY, M., OMRAN, M.H., EL-GARF, W.T. and GOUELI, S.A., 2006. HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro. World Journal of Gastroenterology, vol. 12, no. 30, pp. 4836-4842. PMid:16937465.
• ESGHAEI, M., GHAFFARI, H., RAHIMI ESBOEI, B., EBRAHIMI TAPEH, Z., BOKHARAEI SALIM, F. and MOTEVALIAN, M., 2018. Evaluation of anticancer activity of Camellia sinensis in the Caco-2 colorectal cancer cell line. Asian Pacific journal of cancer prevention. APJCP, vol. 19, no. 6, pp. 1697-1701. PMid:29938468.
• GALVE-ROPERH, I., SÁNCHEZ, C., CORTÉS, M.L., GÓMEZ DEL PULGAR, T., IZQUIERDO, M. and GUZMÁN, M., 2000. Anti-tumoral action of cannabinoids: involvement of sustained ceramide accumulation and extracellular signal-regulated kinase activation. Nature Medicine, vol. 6, no. 3, pp. 313-319. http://dx.doi.org/10.1038/73171 PMid:10700234.
  » http://dx.doi.org/10.1038/73171
• GUARNERI, V. and CONTE, P.F., 2004. The curability of breast cancer and the treatment of advanced disease. European Journal of Nuclear Medicine and Molecular Imaging, vol. 31, no. 0, suppl. 1, pp. S149-S161. http://dx.doi.org/10.1007/s00259-004-1538-5 PMid:15107948.
  » http://dx.doi.org/10.1007/s00259-004-1538-5
• HAMZAOGLU, İ., SARIBEYOGLU, K., DURAK, H., KARAHASANOGLU, T., BAYRAK, İ., ALTUG, T., SIRIN, F. and SARIYAR, M., 2000. Protective covering of surgical wounds with honey impedes tumor implantation. Archives of Surgery, vol. 135, no. 12, pp. 1414-1417. http://dx.doi.org/10.1001/archsurg.135.12.1414 PMid:11115344.
  » http://dx.doi.org/10.1001/archsurg.135.12.1414
• HENDERSON, I.C., BERRY, D.A., DEMETRI, G.D., CIRRINCIONE, C.T., GOLDSTEIN, L.J., MARTINO, S., INGLE, J.N., COOPER, M.R., HAYES, D.F., TKACZUK, K.H., FLEMING, G., HOLLAND, J.F., DUGGAN, D.B., CARPENTER, J.T., FREI 3rd, E., SCHILSKY, R.L., WOOD, W.C., MUSS, H.B. and NORTON, L., 2003. Improved outcomes from adding sequential paclitaxel but not from escalating doxorubicin dose in an adjuvant chemotherapy regimen for patients with node-positive primary breast cancer. Journal of Clinical Oncology, vol. 21, no. 6, pp. 976-983. http://dx.doi.org/10.1200/JCO.2003.02.063 PMid:12637460.
  » http://dx.doi.org/10.1200/JCO.2003.02.063
• HERNANDEZ-LEDESMA, B., HSIEH, C.C. and DE LUMEN, B.O., 2013. Chemopreventive properties of Peptide Lunasin: a review. Protein and Peptide Letters, vol. 20, no. 4, pp. 424-432. PMid:23016582.
• HO, Y.C., YANG, S.F., PENG, C.Y., CHOU, M.Y. and CHANG, Y.C., 2007. Epigallocatechin‐3‐gallate inhibits the invasion of human oral cancer cells and decreases the productions of matrix metalloproteinases and urokinase‐plasminogen activator. Journal of Oral Pathology & Medicine, vol. 36, no. 10, pp. 588-593. http://dx.doi.org/10.1111/j.1600-0714.2007.00588.x PMid:17944751.
  » http://dx.doi.org/10.1111/j.1600-0714.2007.00588.x
• JEMAL, A., BRAY, F., CENTER, M.M., FERLAY, J., WARD, E. and FORMAN, D., 2011. Global cancer statistics. CA: a Cancer Journal for Clinicians, vol. 61, no. 2, pp. 69-90. http://dx.doi.org/10.3322/caac.20107 PMid:21296855.
  » http://dx.doi.org/10.3322/caac.20107
• KIM, E.-Y., KIM, D.-G., KIM, Y.-R., HWANG, H.-J., NAM, T.-J. and KONG, I.-S., 2011. An improved method of protein isolation and proteome analysis with Saccharina japonica (Laminariales) incubated under different pH conditions. Journal of Applied Phycology, vol. 23, no. 1, pp. 123-130. http://dx.doi.org/10.1007/s10811-010-9550-6
  » http://dx.doi.org/10.1007/s10811-010-9550-6
• LI, Z., YANG, L., WANG, J., SHI, W., PAWAR, R.A., LIU, Y., XU, C., CONG, W., HU, Q., LU, T., XIA, F., GUO, W., ZHAO, M. and ZHANG, Y., 2010. β-Actin is a useful internal control for tissue-specific gene expression studies using quantitative real-time PCR in the half-smooth tongue sole Cynoglossus semilaevis challenged with LPS or Vibrio anguillarum. Fish & Shellfish Immunology, vol. 29, no. 1, pp. 89-93. http://dx.doi.org/10.1016/j.fsi.2010.02.021 PMid:20227507.
  » http://dx.doi.org/10.1016/j.fsi.2010.02.021
• LIU, S., LI, Y., CHEN, W., ZHENG, P., LIU, T., HE, W., ZHANG, J. and ZENG, X., 2012. Silencing glypican-3 expression induces apoptosis in human hepatocellular carcinoma cells. Biochemical and Biophysical Research Communications, vol. 419, no. 4, pp. 656-661. http://dx.doi.org/10.1016/j.bbrc.2012.02.069 PMid:22382024.
  » http://dx.doi.org/10.1016/j.bbrc.2012.02.069
• LOGGI, E., VUKOTIC, R. and ANDREONE, P., 2018. Managing HCV treatment failure and the potential of resistance testing in informing second-line therapy options. Expert Review of Anti-Infective Therapy, vol. 16, no. 11, pp. 833-838. http://dx.doi.org/10.1080/14787210.2018.1538783 PMid:30336699.
  » http://dx.doi.org/10.1080/14787210.2018.1538783
• LUSVARGHI, S. and BEWLEY, C.A., 2016. Griffithsin: an antiviral lectin with outstanding therapeutic potential. Viruses, vol. 8, no. 10, pp. 296. http://dx.doi.org/10.3390/v8100296 PMid:27783038.
  » http://dx.doi.org/10.3390/v8100296
• MAILLARD, E., 2011. Epidemiology, natural history and pathogenesis of hepatocellular carcinoma. Cancer Radiotherapie: Journal de la Societe Francaise de Radiotherapie Oncologique, 15, 3-6.
• MANNS, M.P., WEDEMEYER, H. and CORNBERG, M., 2006. Treating viral hepatitis C: efficacy, side effects, and complications. Gut, vol. 55, no. 9, pp. 1350-1359. http://dx.doi.org/10.1136/gut.2005.076646 PMid:16905701.
  » http://dx.doi.org/10.1136/gut.2005.076646
• MOREGREEN, 2017 [viewed in 23 September 2017]. Home seeds [online]. Availavle from: www.moregreen.pk.
• NOREEN, S., HUSSAIN, I., TARIQ, M.I., IJAZ, B., IQBAL, S., QAMAR-UL-ZAMAN., ASHFAQ, U.A. and HUSNAIN, T., 2015. Portulaca oleracea L. as a prospective candidate inhibitor of hepatitis C virus NS3 serine protease. Viral Immunology, vol. 28, no. 5, pp. 282-289. http://dx.doi.org/10.1089/vim.2014.0079 PMid:25871297.
  » http://dx.doi.org/10.1089/vim.2014.0079
• PALUMBO, E., 2011. Pegylated interferon and ribavirin treatment for hepatitis C virus infection. Therapeutic Advances in Chronic Disease, vol. 2, no. 1, pp. 39-45. http://dx.doi.org/10.1177/2040622310384308 PMid:23251740.
  » http://dx.doi.org/10.1177/2040622310384308
• PETERS, G.J., BACKUS, H., FREEMANTLE, S., VAN TRIEST, B., CODACCI-PISANELLI, G., VAN DER WILT, C., SMID, K., LUNEC, J., CALVERT, A., MARSH, S., MCLEOD, H.L., BLOEMENA, E., MEIJER, S., JANSEN, G., VAN GROENINGEN, C.J. and PINEDO, H.M., 2002. Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism. Biochimica et Biophysica Acta, vol. 1587, no. 2-3, pp. 194-205. http://dx.doi.org/10.1016/S0925-4439(02)00082-0 PMid:12084461.
  » http://dx.doi.org/10.1016/S0925-4439(02)00082-0
• PONGTHANAPISITH, V., IKUTA, K., PUTHAVATHANA, P. and LEELAMANIT, W., 2013. Antiviral protein of Momordica charantia L. inhibits different subtypes of Influenza A. Evidence-Based Complementary and Alternative Medicine, vol. 2013, pp. 729081. http://dx.doi.org/10.1155/2013/729081 PMid:23935676.
  » http://dx.doi.org/10.1155/2013/729081
• REHMAN, S., ASHFAQ, U.A., RIAZ, S., JAVED, T. and RIAZUDDIN, S., 2011. Antiviral activity of Acacia nilotica against Hepatitis C Virus in liver infected cells. Virology Journal, vol. 8, pp. 220. http://dx.doi.org/10.1186/1743-422X-8-220 PMid:21569385.
  » http://dx.doi.org/10.1186/1743-422X-8-220
• RIO, D. C., ARES, M., HANNON, G. J. & NILSEN, T. W., 2010. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc, vol. 2010, no. 6, pp. pdb.prot5439. http://dx.doi.org/10.1101/pdb.prot5439 PMID: 20516177.
  » http://dx.doi.org/10.1101/pdb.prot5439
• RUAN, J., LIU, F., CHEN, X., ZHAO, P., SU, N., XIE, G., CHEN, J., ZHENG, D. and LUO, R., 2011. Inhibition of glypican-3 expression via RNA interference influences the growth and invasive ability of the MHCC97-H human hepatocellular carcinoma cell line. International Journal of Molecular Medicine, vol. 28, no. 4, pp. 497-503. PMid:21617840.
• SAEED, A., RAFIQ, Z., SAEED, Q., KHALIQ, B., ULLAH, A., MEHMOOD, S., ALI, Z., ASHRAF, M.Y. and AKREM, A., 2021. Functional characterization of a potent antimicrobial and insecticidal chitin binding protein from seeds of Iberis Umbellata L. Pakistan Journal of Botany, vol. 53, no. 4, pp. 1515-1523. http://dx.doi.org/10.30848/PJB2021-4(16)
  » http://dx.doi.org/10.30848/PJB2021-4(16)
• SANDSTRÖM, C., BERTEAU, O., GEMMA, E., OSCARSON, S., KENNE, L. and GRONENBORN, A.M., 2004. Atomic mapping of the interactions between the antiviral agent cyanovirin-N and oligomannosides by saturation-transfer difference NMR. Biochemistry, vol. 43, no. 44, pp. 13926-13931. http://dx.doi.org/10.1021/bi048676k PMid:15518540.
  » http://dx.doi.org/10.1021/bi048676k
• SA-NGIAMSUNTORN, K., WONGKAJORNSILP, A., PHANTHONG, P., BORWORNPINYO, S., KITIYANANT, N., CHANTRATITA, W. and HONGENG, S., 2016. A robust model of natural hepatitis C infection using hepatocyte-like cells derived from human induced pluripotent stem cells as a long-term host. Virology Journal, vol. 13, no. 1, pp. 1-18. http://dx.doi.org/10.1186/s12985-016-0519-1 PMid:27044429.
  » http://dx.doi.org/10.1186/s12985-016-0519-1
• SCOTT, L.J., 2018. Ledipasvir/sofosbuvir: a review in chronic hepatitis C. Drugs, vol. 78, no. 2, pp. 245-256. http://dx.doi.org/10.1007/s40265-018-0864-z PMid:29380288.
  » http://dx.doi.org/10.1007/s40265-018-0864-z
• SENTHILRAJA, P. and KATHIRESAN, K., 2015. In vitro cytotoxicity MTT assay in Vero, HepG2 and MCF-7 cell lines study of Marine Yeast. Journal of Applied Pharmaceutical Science, vol. 5, pp. 80-84. http://dx.doi.org/10.7324/JAPS.2015.50313
  » http://dx.doi.org/10.7324/JAPS.2015.50313
• SHAIKH, A.Y. and SHIH, J.A., 2012. Chemotherapy-induced cardiotoxicity. Current Heart Failure Reports, vol. 9, no. 2, pp. 117-127. http://dx.doi.org/10.1007/s11897-012-0083-y PMid:22382639.
  » http://dx.doi.org/10.1007/s11897-012-0083-y
• SHIVAMADHU, M.C., SRINIVAS, B.K., JAYARAMA, S. and CHANDRASHEKARAIAH, S.A., 2017. Anti-cancer and anti-angiogenic effects of partially purified lectin from Praecitrullus fistulosus fruit on in vitro and in vivo model. Biomedicine and Pharmacotherapy, vol. 96, pp. 1299-1309. http://dx.doi.org/10.1016/j.biopha.2017.11.082 PMid:29174033.
  » http://dx.doi.org/10.1016/j.biopha.2017.11.082
• SMITH, B., 1984. SDS polyacrylamide gel electrophoresis of proteins. Proteins. Springer. http://dx.doi.org/10.1385/0-89603-062-8:41
  » http://dx.doi.org/10.1385/0-89603-062-8:41
• SUN, C.K., CHUA, M.S., HE, J. and SO, S.K., 2011. Suppression of glypican 3 inhibits growth of hepatocellular carcinoma cells through up-regulation of TGF-β2. Neoplasia (New York, N.Y.), vol. 13, no. 8, pp. 735-47. http://dx.doi.org/10.1593/neo.11664 PMid:21847365.
  » http://dx.doi.org/10.1593/neo.11664
• TANG, H., TANG, X.-Y., LIU, M. and LI, X., 2008. Targeting alpha-fetoprotein represses the proliferation of hepatoma cells via regulation of the cell cycle. Clinica Chimica Acta, vol. 394, no. 1-2, pp. 81-88. http://dx.doi.org/10.1016/j.cca.2008.04.012 PMid:18485897.
  » http://dx.doi.org/10.1016/j.cca.2008.04.012
• THOMAS, A.S., SARAVANAKUMAR, R. and GUPTA, P.V., 2017. Evaluation of cytotoxic activity of protein extracts from the leaves of Morinda pubescens on human cancer cell lines. Revista Brasileira de Farmacognosia, vol. 27, no. 1, pp. 99-104. http://dx.doi.org/10.1016/j.bjp.2016.08.003
  » http://dx.doi.org/10.1016/j.bjp.2016.08.003
• UMER, M. and IQBAL, M., 2016. Hepatitis C virus prevalence and genotype distribution in Pakistan: comprehensive review of recent data. World Journal of Gastroenterology, vol. 22, no. 4, pp. 1684-1700. http://dx.doi.org/10.3748/wjg.v22.i4.1684 PMid:26819533.
  » http://dx.doi.org/10.3748/wjg.v22.i4.1684
• VIJAYAN, P., RAGHU, C., ASHOK, G., DHANARAJ, S. and SURESH, B., 2004. Antiviral activity of medicinal plants of Nilgiris. The Indian Journal of Medical Research, vol. 120, no. 1, pp. 24-29. PMid:15299228.
• WANG, J., ITO, H. and SHIMURA, K., 1989. Enhancing effect of antitumor polysaccharide from Astragalus or Radix hedysarum on C3 cleavage production of macrophages in mice. Japanese Journal of Pharmacology, vol. 51, no. 3, pp. 432-434. http://dx.doi.org/10.1016/S0021-5198(19)40106-6 PMid:2622096.
  » http://dx.doi.org/10.1016/S0021-5198(19)40106-6
• WEIDNER, C., ROUSSEAU, M., PLAUTH, A., WOWRO, S.J., FISCHER, C., ABDEL-AZIZ, H. and SAUER, S., 2016. Iberis amara extract induces intracellular formation of reactive oxygen species and inhibits colon cancer. PLoS One, vol. 11, no. 4, pp. e0152398. http://dx.doi.org/10.1371/journal.pone.0152398 PMid:27050665.
  » http://dx.doi.org/10.1371/journal.pone.0152398
• YANG, J.D. and ROBERTS, L.R., 2010. Hepatocellular carcinoma: a global view. Nature Reviews. Gastroenterology & Hepatology, vol. 7, no. 8, pp. 448-458. http://dx.doi.org/10.1038/nrgastro.2010.100 PMid:20628345.
  » http://dx.doi.org/10.1038/nrgastro.2010.100
• ZEKRI, A.R.N., BAHNASSY, A.A., EL-DIN, H.M.A. and SALAMA, H.M., 2009. Consensus siRNA for inhibition of HCV genotype-4 replication. Virology Journal, vol. 6, pp. 13. http://dx.doi.org/10.1186/1743-422X-6-13 PMid:19173711.
  » http://dx.doi.org/10.1186/1743-422X-6-13
• ZHU, F., YUAN, S., ZHANG, Z.-W., QIAN, K., FENG, J.-G. and YANG, Y.-Z., 2016. Pokeweed antiviral protein (PAP) increases plant systemic resistance to Tobacco mosaic virus infection in Nicotiana benthamiana. European Journal of Plant Pathology, vol. 146, no. 3, pp. 541-549. http://dx.doi.org/10.1007/s10658-016-0938-2
  » http://dx.doi.org/10.1007/s10658-016-0938-2
• WORLD HEALTH ORGANIZATION - WHO, 2017 [viewed in 10 December 2017]. Global hepatitis report 2017 executive summary: World Health Organization [online]. Available from: https://www.who.int/publications/i/item/global-hepatitis-report-2017
  » https://www.who.int/publications/i/item/global-hepatitis-report-2017

Publication Dates

• Publication in this collection
  01 Apr 2022
• Date of issue
  2024

History

• Received
  27 May 2021
• Accepted
  02 Jan 2022