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Year : 2019  |  Volume : 15  |  Issue : 1  |  Page : 176-184

The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line

1 Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran
2 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran

Date of Web Publication13-Mar-2019

Correspondence Address:
Dr. Mahsa Alem
Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, East Azerbaijan
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_41_17

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 > Abstract 

Context: Tilorone dihydrochloride is a therapeutic agent with a different mechanism in cancer. The species of Lactobacillus have an important role in cytotoxic effect.
Aims: Because of unknown effects of tilorone and culture supernatants from Lactobacillus reuteri on hepatoma, the aim of this study is to evaluate apoptotic, cytotoxic, and therapeutic effects of tilorone on mouse hepatoma cell line with and without culture supernatants from L. reuteri.
Materials and Methods: To do so, after cell line culture, cells were divided into different groups such as negative control, treatment with four doses of tilorone, positive control of supernatant (single dose), and combination therapy groups of different doses of tilorone with supernatant (constant doses), for 48 h. All groups were studied with pathologic tests, biochemical study, tetrazolium dye (3-(4, 5- dimethylthiazol -2-yl)-2, 5-diphenyltetrazolium bromide [MTT]) assay, and absolute real-time-polymerase chain reaction (RT-PCR) were done to assess Bax and Bcl-2 genes expression, as molecular studies.
Results: MTT assay results revealed that the tilorone tissue culture IC50 (TCIC50) on the Hepa1-6 cell line was 50 μg/ml. RT-PCR analysis showed that tilorone dihydrochloride induced upregulation and downregulation in expression of Bax and Bcl-2, respectively. Simultaneous, antioxidant effect has also seen in a way that prevented necrosis, in biochemical analysis. These results were dose dependent and statistically significant compared to the control group.
Conclusions: Based on these results, it appeared that this agent could be a good candidate for further evaluation as effective chemotherapy acting through the induction of apoptosis in hepatoma. The cell death caused through bacterial supernatant was rather necrosis than apoptosis.

Keywords: Bax and Bcl-2 genes, cytotoxic, hepatoma cell line, tilorone dihydrochloride

How to cite this article:
Alem M, Shahbazfar AA, Zare P, Tayefi-Nasrabadi H. The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line. J Can Res Ther 2019;15:176-84

How to cite this URL:
Alem M, Shahbazfar AA, Zare P, Tayefi-Nasrabadi H. The effects of coadministration of tilorone dihydrochloride and culture supernatants from Lactobacillus reuteri on the mouse hepatoma cell line. J Can Res Ther [serial online] 2019 [cited 2021 Jun 20];15:176-84. Available from: https://www.cancerjournal.net/text.asp?2019/15/1/176/230444

 > Introduction Top

Liver cancer has become a worldwide concern and it is the third cause of cancer-related mortalities worldwide. According to some references, hepatoma is currently the fifth deadliest cancer worldwide with bad prognosis and poor diagnosis. However, within other research, hepatoma is one of the prevalent global malignancies.[1] Because of rapid tumor progression, recurrence, and drug-resistance of hepatocellular carcinoma, despite the common clinically treatment, its mortality still remains high. Development of exquisite strategies and targeting therapeutic methods for hepatoma is highly preferred.[2]

Various biologic markers and factors determine the recurrence of neoplasia. Proliferative activity and chaos of apoptosis of hepatic cells have been related to significant factors for hepatoma progression and recurrence. Overexpression of Bax is transmitted to the mitochondria, followed by it the cytochrome c is released, and conjugates with Apaf-1 and caspase-9, subsequently, apoptosis induction occurs in cells through the internal pathway. Despite Bax, Bcl-2 can constitute heterodimers with Bax, thereupon inhibits Bax activity. Unlike Bcl-2, Bax gene expression is directly associated with apoptosis and has a negative correlation with the proliferative activity of tumor cells. Therefore, the ratio of Bcl-2 to Bax is correlated with apoptosis in neoplastic cells.[3]

The Hepa1-6 cell line is a suitable model for studying liver cancer.[4] We used the Hepa1-6 (mouse hepatoma) cells for determining the antitumor efficacy and inhibitory ability of tilorone and  Lactobacillus reuteri Scientific Name Search o.

Tilorone Dihydrochloride is a fluorenone-based compound that activates as antiviral[5] agent and treats cancer.[6] In addition, this agent has immunomodulatory activities such as significantly increasing interferon and interferon inducers[7] and enhanced phagocytic activity of natural killer cell activation. Other pharmacological functions of this agent are anti-inflammatory, antipyretic, and antifibrotic.[6] The amine group and amine ring of tilorone have a specific site on deoxyribonucleic acid (DNA) and by sitting between its 2 strands leads to therapeutic effect.[8] The other mechanisms of its effects are inhibition of expression of cyclin-dependent Kinase gene,[9] chromatin structure regulating and involvement in gene expression,[5] and interference in signaling pathways and transcription factors.[10] In the study, its effects on the reticuloendothelial system,and the immune response were clarified.[11] On the other hand, Low concentrations of tilorone induced lysosomal deposits of glycosaminoglycans sulfate in vivo and in vitro.[12],[13],[14] Tilorone binds to DNA and this DNA strand interaction inhibits the DNA template functions in DNA- and RNA polymerase reactions in vitro and in RNA tumor viruses.[8] For the exact mechanisms of action of tilorone, more studies are still needed.

Cytotoxic and therapeutic properties of tilorone and its derivatives have been shown in many in vivo and in vitro experiments;[8] however, there are not any investigation on its combination with the bacterial supernatant.

In recent years, a wide variety of bacterial targeting cancer therapy has been investigated in clinical and preclinical experiments.[15] Studies have shown that secondary metabolites of bacteria possess biological activity. These properties include anti-inflammatory and anti-oxidative effects.[16] The species of Lactobacillus, one of the intestinal floras, have the largest usage in biotechnology. The bacteria especially Lactobacillus have useful effects on stasis of different cancerous blood cell lines.[17],[18] The most important effective productions of them are lactic acid, exopolysaccharide, biosurfactants, peptidase, and the effective material in low quantity are hydrogen peroxide, acetic acid, formic acid, and diacetyl. They have an important role in regulation and conduction of cell function and cytotoxic effect, directly, indirectly, and competitively.[19] Recently, using bacteria as alternative cancer therapeutics has been explored.[20],[21] For example,  Salmonella More Details enterica serovar typhimurium SL7207 showed promising therapeutic potential for solid tumor such as breast, colon, lung, prostate cancer, neuroblastoma, fibrosarcoma, and hepatoma in preclinical experiments.[15] Some studies clarified that butyric acid, a product of bacterial anaerobic metabolism, induces apoptosis and inhibits cell growth in a broad spectrum of cancerous cell lines.[22] In addition, Singh and Lai in 2005 demonstrated that sodium butyrate yield cell death on Molt-4 cell line as a model for human lymphoblastoid leukemia.[23] Among these bacteria, L. reuteri is a potential election to cause advantageous therapeutic consequences and fewer side effects.[17] L. reuteri strains could ferment glycerol and produce a strong and broad-spectrum antibiotics called reuterin that inhibit the growth of Gram-positive and Gram-negative bacteria, yeasts, and protozoa.[24] Although the effectiveness of Lactobacillus components on cell death and proliferation has been proven, its usage in hepatoma is still unknown, yet its impact is completely understood.

In the current study, the addition of L. reuteri to the combination was done to enhance the cytotoxic potency of tilorone. We proposed that tilorone and L. reuteri might have safely and effectively inhibit hepatoma tumor growth and might be a candidate for development as a therapeutic agent that treatment cancer. On the other hand, the relation between tilorone effects and expression of apoptosis-related genes on hepatoma cell line was investigated.

 > Materials and Methods Top

Cell culture

The Hepa1-6 cell line (ATCC: CRL-1830) was obtained from National Cell Bank of Iran (Pasteur Institute, Iran). This cell line is adherent, epithelial-like cells growing as monolayers widely used as a well-known model for hepatocarcinoma in cytotoxic research projects.[4] The cells were cultivated in a monolayer culture in 25 cm2 T-flasks in Roswell Park Memorial Institute 1640 (Gibco, USA), (enriched with L-glutamine) culture medium, without antibiotics, supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), at 37°C in a 5% CO2 humidified atmosphere. Cells were trypsinized and subcultured routinely when they reached almost 80% confluence. Then cells were seeded into 96-well plates at a density of 10,000 cells/well for 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and into 6-well plates at a density of 0.5 × 104 for gene expression and apoptosis analysis. All procedures were performed under the Class II laminar hood for cell culture.

Bacterial culture and extraction of bacterial secondary metabolites

L. reuteri (With the reference number DSM 20079, PTCC 1655, ATCC 4356) was purchased from the Iran's industrial collection center of fungi and bacteria, as lyophilized vials. According to the provided instructions, the bacteria were activated. For bacterial manipulations and maintenance, bacteria were grown at 37°C in 400 μl of liquid de Man-Rogosa and Sharpe broth (Merck Millipore, Germany) medium culture using standard procedures. All procedures were performed under the Class II laminar hood. Media-containing L. reuteri in the anaerobic gas pack (Anaerocult) were incubated for 48 h at 37°C. To obtain a supernatant free of bacteria (cell-free supernatant) as a treatment, bacteria were isolated by centrifugation. NaOH (one normal) was used for neutralizing the supernatant at 4.22 pH. Otherwise, this acidity can obstacle to growth or even lead to cell line death. Finally, the supernatant was filtered with cellulose acetate single usage syringe filters with a pore diameter of 0.22 μm (GyroDisc, Orange Scientific, Belgium), then it was frozen and stored at −20°C temperature until usage.[25],[26]


Tilorone dihydrochloride with molecular weight of 483.4709 (purum ≥98%) were purchased from Sigma-Aldrich Corp, USA. IUPAC name of tilorone dihydrochloride is 2,7-Bis (2-[diethylamino] ethoxy)-9-fluorenone dihydrochloride and the empirical formula is C25H36 Cl2N2O3.

According to agent preparation, tilorone dissolved in dimethyl sulfoxide (DMSO) (Merck Millipore, Germany) at a concentration of 10 mM (1 mg/2 ml of DMSO) as a stock solution and stored at room temperature until use. Each stock solution was serially diluted in the medium at each well of plates so that most final concentration of DMSO in the culture cell did not exceed 0.1%. Different doses of 99% pure tilorone (1, 10, 50, and 100 μg/ml) were added to the culture media.

To study the effects of tilorone on the growth of Hepa1-6, the treated series were incubated at the desired concentration of tilorone for 48 h and afterward cells were fed with the fresh normal medium.


The cultivated cell line was classified into 10 groups as follows:

  1. Investigate the effects of the first dose of the drug (1 μg/ml) on the cell line (2.1 μl/ml of Stoke complete environment) (T1)
  2. Investigate the effects of the second dose of drug (10 μg/ml) on the cell line (21 μl/ml of Stoke complete environment) (T2)
  3. Investigate the effects of the third dose of drug (50 μg/ml) on the cell line (105 μl/ml of Stoke complete environment) (T3)
  4. Investigate the effects of the fourth dose of drug (100 μg/ml) on the cell line (210 μl/ml of Stoke complete environment) (T4)
  5. Negative control (C)
  6. Investigate the effects of the constant dose of bacterial supernatant (10 μl/ml) on medium with the first dose of drug (T1 + S)
  7. Investigate the effects of the constant dose of bacterial supernatant (10 μl/ml) on medium with the second dose of the drug (T2 + S)
  8. Investigate the effects of the constant dose of bacterial supernatant (10 μl/ml) on medium with the third dose of the drug (T3 + S)
  9. Investigate the effects of the constant dose of bacterial supernatant (10 μl/ml) on medium with the fourth dose of the drug (T4 + S)
  10. Investigate the effect of the bacterial supernatant alone constant dose (10 μl/ml) on the cell line (S).

Exposure time at any test was 48 h.

Biochemical analysis

Cells were incubated for 48 h; subsequently for precipitating the floating cells, harvesting supernatant centrifuged at 5000 rpm for 5 min. The superior fluid was used as a supernatant or medium culture for biochemical examination. To prepare cell fraction or cell lysate extracts in the first step, the cells in the bottom of the flask were scraped through a stirring rod. The second step for this purpose is repeating the cycles of freeze-thawing for the scraped cells and the centrifuged precipitate. In the third step, this extract was centrifuged to precipitate cleaved cells. Finally, the cell fraction extract was used for measuring the total protein, lactate dehydrogenase (LDH), and lipid peroxidation product.[27]

One of the cell damage indicators is LDH. Babson and Babson's method was utilized for measuring LDH activity.[28] This test is based on the conversion of pyruvate to lactate and the reduced form of nicotinamide adenine dinucleotide (NADH) to the oxidized form of nicotinamide adenine dinucleotide (NAD+) in the presence of LDH. In this test, with higher amount of LDH, NADH turns more quickly to NAD+.[29] By measuring the enzyme changes, according to the total protein, we were able to interpret the changes. Total protein was measured by the Lowry method separately inhomogeneous liquid cell fraction, culture medium, and supernatant of cell fraction. In this method, bovine serum albumin was used as a standard. This method is based on the peptide linkage with bivalent copper ions (Cu2+) to form a complex that increases the absorption at a wavelength of 550 nm in the presence of the Folin–Ciocalteu reagent.[30]

During cell damage, cell membrane unsaturated fatty acids react with free radicals and malonyl dialdehyde (MDA) is produced as a result. Hence, to measure the standard indicator of the lipid peroxidation, MDA was evaluated through the thiobarbituric acid reactive substance method.[31] This measurement is proposed as nonenzymatic biomarkers of oxidative stress. Whenever the oxidative stress occurs, lipid peroxidation is increased. At this time, MDA reacts with the thiobarbituric acid in acidic pH and high temperature. The pink complex has a maximum absorbance at 532 nm.[27],[32]

Pathologic analysis

At the end of the study, the cellular deviations were investigated and photographed under an inverted microscope every 12 h. After throwing out the contents of each well, the remaining cells on the 6-well plates were fixed with 100 μl/well methanol for 3 min and thrown out again and dried, then stained with H and E and were monitored under a light microscope. For staining, at first, 3 ml hematoxylin were added to each well for 1.5 min, then it was thrown out, and plates were washed by flowing water. Eosin staining was carried out with the same technique for 3 min.[27],[33]

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

MTT reagent (3-(4, 5-dimetylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (M5655) Thiazolyl Blue Tetrazolium Bromide 98% was purchased from Sigma-Aldrich Corp, USA. For MTT assay, the cells were seeded at 5 × 103 cells per well in 96-well plates and cultured at 37°C for 24 h, then cells in the exponential phase of growth were exposed to different concentrations of Tilorone (1-100 μg/ml). The concentration of drug that repressed cell proliferation by 50% (IC50s) was determined from MTT assay data after 48 h treatments.[34]

Bax and Bcl-2 genes expression

The cells were seeded in 10 flasks with a concentration of 1 × 106 cell/flask. Then, three flasks were treated with IC50 48 h at 48 h, and the last flask was as a control group. Total messenger RNA (mRNA) extraction was done from cultured cells using commercial kits of intron's company, according to the manufacturer's protocols and 2 μl of total RNA was applied for 1 step and absolute real-time-polymerase chain reaction (RT-PCR). Quantification of genes expression was performed using a master mix from Bioneer-AccuPower Rocket Script RT quantitative reverse transcription PCR (RT-qPCR) PreMix Kit (including Taq DNA polymerase and TaqMan probe). Briefly, 1 μl of a forward primer and 1 μl of reverse primer, 5 μl of Master-mix, and 7 μl of nuclease free water were subjected to PCR-amplification reaction.

Bioneer company primer is Taq labeled along with fluorescent molecules and probes (TaqMan probe method). This probe prevents the weak reaction and being primer-dimer of nucleotides. TaqMan techniques used in this study for RT-PCR, poses more sensitivity, specificity, and repeatability, compared to SYBR Green. This difference related to the use of the third oligonucleotide is labeled with a fluorescent stain. The TaqMan probes had been labeled with a reporter fluorescent stain at the 5' end and a quencher fluorescent stain at the 3' end. Whenever the probe was intact, any signal was not emitted. Nuclease degradation of the hybridization probe eliminated the quenching influence of TAMRA. On the other hand, the TaqMan method is preferred due to biosafety rules for users and the environment. Therefore, mutagenic ethidium bromide is not used in this method. In this technique, a high amount of sample complementary DNA (cDNA), more replication, and more intensity of fluorescence radiation is immediately registered by the device and vice versa.[35] In this study, RT-PCR with absolute quantification was done.[36] At first, reverse transcription step was done at 48°C for 15 min for cDNA synthesis (1 cycle). The thermocycling conditions of PCR reactions were initiated at 95°C for 5 min (1 cycle) as a predenaturation step, followed by 40 PCR cycles and in recognition of a denaturation step at 95°C for 30 s, and annealing/extension/detection at 60°C for 35 s.

For amplification of Bax and Bcl-2 cDNA, primers and probes were designed and temperature and period were set on to QIAGEN-five-channel - thermo-cycler (Rotor-Gene Q 6000- Germany). Designed TaqMan primers were 5′-TGATCAGAACCATCATG-3′ (forward) and 5′-GTTGACCAGAGTGGCGTAGG-3′ (reverse) for Bax and 5′-TCGCAGAGATGTCCAGTCAG-3′ (forward) and 5′-ATGCCGGTTCAGGTACTCAG-3′ (reverse) for Bcl-2. Gene expression levels and the mRNA copy number of each sample were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH+) gene (as a housekeeping control gene) (Forward primer: 5′-GACCACAGTCCATGCCATCAC-3′ and Reverse-primer: 5′-GCTGTTGAAGTCGCAGGAGAC-3′). The specificity of the primers and the purity of the final PCR product delineated by performing melting curve at the end of each PCR cycle. To obtain normalized amounts of transcripts, the copy number of Bcl-2 and Bax were divided through the amount of GAPDH mRNA for each sample.[35],[37]

Statistical analysis

The amount of Bax and Bcl-2 genes expression changes were analyzed by Rest software, and MTT and biochemical data were analyzed through version 21.0 of SPSS (Statistical Package for Social Sciences, SPSS software 21, IBM Inc., NY, the USA). Statistical analysis was performed using one-way analysis of variance (ANOVA test) to compare the quantitative data. Each one of the groups was compared by Tukey test as post hoc. Values were presented as mean ± standard error of the mean and P < 0.05 was considered as statistically significant. The data of this present study were resulted from at least three independent experiments.

 > Results Top

The accuracy and reproducibility of each experiment's results were proved through repeating and forming triplicate manner.

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

Tilorone induces DNA damage, and therefore, it can impose an antiproliferative effect on the Hepa1-6 cells. After treatment of the Hepa1-6 cell line with different concentrations of tilorone, tilorone TCIC50 (Tissue Culture IC50) and changes in the cell life and death was determined through MTT. Treatment period was set to 48 h to permit adequate time for arriving at the cycle of cells. The results of MTT assay after 48 h treatment revealed that 50 μg/ml tilorone dihydrochloride causes cell death in half of cellular population (TCIC50 of 48 h) [Table 1] and [Figure 1].
Table 1: The effects of tilorone dihydrochloride and Lactobacillus reuteri supernatant on Bax and Bcl-2 gene expression, the results of 3-(4, 5-dimetylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, biochemical analysis of lactate dehydrogenase and malonyl dialdehyde in cell culture medium and cell fraction of the hepa1-6 cell line

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Figure 1: The effects of tilorone dihydrochloride the results of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and the cell viability of Hepa1-6 cell line

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Induced morphological changes were studied using H and E staining and light microscopy on the Hepa1-6 cells treated with tilorone dihydrochloride and the supernatant of L. reuteri culture.

In the six groups treated with different doses of tilorone, without and with bacterial supernatant, dose-dependent pathological changes were observed. These changes included cell swelling and vacuolation. After 48 h of exposure, the control groups did not show any specific change with full cell population [Figure 2]. The group treated with a lower dose of tilorone [Figure 3] and [Figure 4] showed identical changes, with less severity, compared to the group that received the highest dose of it [Figure 5]. This change includes mild cell swelling and few cases of vacuolation. The highest dose of tilorone caused all of the cells' detachment from the flask bottom, in the Groups 4, 9, and 8. The addition of bacterial supernatant in constant dose with the combination therapy caused more pathological changes and cell death [Figure 6], [Figure 7], [Figure 8] compared to [Figure 3], [Figure 4], [Figure 5], respectively]. The group treated with bacterial supernatant alone, showed vacuolation in the cytoplasm of cells in this group [Figure 9].
Figure 2: Control group did not show any specific change with full cell population in Group 5; H and E; light microscopy; Hour 48 × 200

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Figure 3: Fine vacuolation (white arrow) in the cytoplasm and mild swelling of few cells in Group 1 (Tilorone 1 μg/ml); H and E; light microscopy; Hour 48 × 400

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Figure 4: Vacuolation (white arrow) in the cytoplasm and cell swelling of Hepa1-6 cells in Group 2 (Tilorone 10 μg/ml); H and E; light microscopy; Hour 48 × 800

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Figure 5: The more amount and small size of vacuolation in the cytoplasm and swelling of few cells in Group 3 (Tilorone 50 μg/ml); H and E; light microscopy; Hour 48 × 800

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Figure 6: Fine vacuolation in the cytoplasm and mild swelling (white arrow) of few cells in Group 6 (Tilorone 1 μg/ml + bacterial supernatant 10 μg/ml); H and E; light microscopy; Hour 48 × 800

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Figure 7: Cytoplasm with significant vacuolation, severe cell swelling, and detachment of some cells from the flask's bottom as hollow cavity (white arrow) in Group 7 (Tilorone 10 μg/ml + Bacterial supernatant 10 μg/ml); H and E; light microscopy; Hour 48 × 800

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Figure 8: Cytoplasm with significant and more vacuolation, cell wrinkling, detachment of cells from the flask's bottom as hollow cavity and cell population decreased exquisitely in Group 8 (Tilorone 50 μg/ml + Supernatant 10 μg/ml); H and E; light microscopy; Hour 48 × 800

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Figure 9: Vacuolation in the cytoplasm of cells in Group 10 (Bacterial supernatant 10 μg/ml); H and E; light microscopy; Hour 48 × 800

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Biochemical results obtained from the culture medium LDH measurements showed that the higher the dose of tilorone, the more LDH activity was seen either in combination therapy groups with tilorone and bacterial supernatant or in monotherapy groups with tilorone, compared to the control group. So that in the four groups only receiving tilorone, the medium LDH level increased significantly in a dose-dependent manner (P < 0.01). The addition of bacterial supernatant to tilorone therapy caused a significant increase of LDH levels in the cell medium supernatant (P = 0.03). In addition, there was a significant increase in the cell fraction LDH (P < 0.01) levels but not in the cell fraction MDA levels. Fraction LDH level in groups that treated with tilorone though indicated significantly increase compared to the control group, but this increasing inversely related to a therapeutic dose. This result seems logical, according to the explanations provided in the discussion. The addition of bacterial supernatant caused a statistically significant decrease in concentration of the cell fraction LDH (P = 0.025). The addition of bacterial supernatant to tilorone therapy did not cause a significant change in MDA levels in the cell fraction extracts [Table 1].

Bax and Bcl-2 gene expression

As it can be seen in [Table 1], in tilorone dihydrochloride-induced toxicity significantly induced Bax gene expression level as pro-apoptotic markers and significantly decreased Bcl-2 gene expression level as anti-apoptotic compared to the control group were observed (P < 0.05). Bax gene expression in the final concentration of tilorone dihydrochloride is more than its primary concentration and had the dose-dependent manner. Thus, the concentration of 10 μg/ml had been more effective than the concentration of 1 μg/ml. On the other hand, the concentration of 100 μg/ml had the highest expression of Bax gene, which means a further process of apoptosis is the fourth concentration (P < 0.05). In the group treated with bacterial supernatant of L. reuteri as well as the control group, there was no expression of Bax gene but in this group, the mRNA level of Bcl2 significantly decreased compared to the control group (P = 0.01). Among four groups that received combination therapy, a little Bax gene expression was observed only in Group 6 (1 μg/ml of tilorone + 10 μg/ml of bacterial supernatant). As it was observed and expected, in all groups Bcl2 gene expression was significantly lower than the control group (P < 0.05). Especially on the first dose of tilorone (Group 1) which was almost not expression in Bcl-2 gene. Therefore, the higher concentration of tilorone treatment induced upregulation of Bax gene expression and downregulation Bcl-2 gene expression more efficiently that caused apoptosis induction and then initiated internal apoptosis (mitochondrial) pathway.

 > Discussion Top

Tilorone dihydrochloride is a therapeutic agent in the treatment of cancers and has a good inhibitory effect on solid tumors such as prostate cancer, laryngeal papillomatosis, cerebral gliomas and metastatic breast cancer.[9] In recent years, Zhou et al. reported tilorone dihydrochloride as new tilorone analogs associated with boosted antitumor activity.[38] This recent study supported our results. The results of our study declared that tilorone dihydrochloride had the beneficial antiproliferative properties on the Hepa1-6 cell line that repressed immortality of this cancer cells. It means that tilorone dihydrochloride might have the therapeutic effects on liver cancer. It has been shown that tilorone dihydrochloride had an antitumor and antimetastatic effect through inhibition of cdk5 in prostatic cancer cell line.[9] In the Hepa1-6 liver cancer cell line, tilorone dihydrochloride caused induction of Bax gene expression and reduction of Bcl-2 gene expression. So that, in higher tilorone dihydrochloride concentrations the expression of Bax gene induced and Bcl-2 gene expression decreased significantly in comparison to control group. Therefore, tilorone dihydrochloride could play an antiproliferative effect on the Hepa1-6 cells and can induce apoptosis in this cell line and it is because of DNA damage induction and activation of internal apoptosis pathway. Although this activity should be supposed the antioxidant effect for tilorone dihydrochloride. Furthermore, this agent probably had the safe effect and reduces the rate of necrosis and low cytotoxicity toward normal cells. This interpretation was also shown in other research.[8] As mentioned earlier, decrease in the level of MDA in therapeutic tilorone group compared to the cancerous control group confirmed this claim. The increment of novel therapeutic strategies which are efficient, positive and subsidiary but less toxic is of high excellence and priority.[39] Although, LDH levels faced an increase. With cell destruction, cell membrane and the membrane of intracellular organelles is permeable to macromolecules such as enzymes, thus LDH enzyme leaks outside of the cell. The more the cell injury, the higher the level of this enzyme is leaked out the medium. Pathological increase in the levels of this enzyme is a marker of the degradation and cell death in evaluated cultured cells. Hence, this test is useful along with other diagnostic tests. Thus, the results of this study suggested the further the dose of tilorone, the more the medium LDH and the less fraction LDH or intracellular LDH were seen. As a result that was leaked out the culture medium.[29] In addition to the leakage a certain enzyme that occurs in the process of necrosis, according to the studies of scientists, even also in the process of apoptosis a degree of enzyme leakage from apoptosome was also observed.[40],[41] The obtained results from this research delineated this issue, too.

The results of the present study declared that tilorone dihydrochloride had a significant histopathological effect on the Hepa1-6 vital parameters.

In the groups that received bacterial supernatant, necrosis was predominantly observed, and in the combination therapy group, apoptosis with a dominance of necrosis was seen. Hence, the addition of L. reuteri could increase the cytopathic effects of tilorone dihydrochloride against the Hepa1-6 cancer cell lines.

Baldwin et al. showed that lactic acid forming probiotic bacteria along with 5 fluorouracil had dose-dependent cytotoxic on colon cancer. This effect related to fast activation of caspase 3 and subsequently enhancement of apoptotic activity, so they antedated that probiotics have cocancer therapy usage, in the future.[42] Some studies demonstrated that Lactobacillus bacteria have a potential role in cancer prevention and treatment. This prevention is related to the reduction of the colonization of cancer cells,[43] induction of apoptosis in antigen-stimulated T cells through modulating dendritic cell function by Lactobacillus acidophilus,[44] and apoptotic induction through the external (not mitochondrial) pathway in gastric cancer cells.[26] Puertollano et al.'s study in 2013 clarified that the culture supernatants from Lactobacillus Plantarum- induced necrosis rather than apoptosis on human promyelocytic leukemia-60 cell line as human promyelocytic leukemia which was in agreement with our data in this study. They used Hoechst 33342 nuclear staining and annexin V/propidium iodide double staining. However, the study of Iyer et al. in 2008 was in disagreement with our results. Because they reported tumor necrosis factor-induced apoptosis effect of L. reuteri on human myeloid leukemia-derived cells through the modulation of nuclear factor-κB and mitogen-activated protein kinase signaling.[17] Except for Lactobacillus species, other probiotic bacteria such as Bifidobacterium Longum was found with antitumor and antimutagenic potential on the colon cancer cell line.[45]

Besides probiotic bacteria, Babu et al. demonstrated that Salmonella typhimurium arrested chicken macrophage cell line and signs of apoptosis were visible.[46] In one of the studies that were done in 2002, Ikeguchi et al. concluded that S. typhimurium SL7207-lux treatment reduced cell viability of human hepatoma HepG2 cells through apoptosis through entry into the cells and localize in the cytoplasm. They used the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL assay to determine this effect and caspase-3 activity assay for confirmation. Inhibition of the proliferation of HepG2 cells was determined by Sulforhodamine B assay.[35] Like this study, conversion expression of Bax-Bcl-2 has been shown in various cancer types.[3],[34]

About the technique used, in 2002 Ikeguchi et al. showed that the qRT-PCR method emerges to be a helpful technique for detection dependable gene expression in cells and this research clarified Bcl-2/Bax mRNA expression ratio to be correlated with tumor cell apoptosis or tumor cell proliferative activity.[35]

 > Conclusion Top

At last, based on the present results, it is concluded that tilorone dihydrochloride induced cell's apoptosis and it acted as an inducer for Bax gene expression and decreased Bcl-2 gene expression. Simultaneous antioxidant effects have been even seen in a way that prevents necrosis, compared to the control group. Altogether, tilorone in the Hepa1-6 cell line induces cancerous cell death and therefore it can be considered for more investigation in animal models, and it could be a good and valuable candidate in hepatoma therapy. On the other hand, a monotherapy with tilorone dihydrochloride alone can be more effective against cancer cells than combination therapy with L. reuteri supernatant, since this supernatant was not effective on hepatoma cancer cells and deviated the nature of cell death from apoptosis to necrosis so we did not find L. reuteri to be effective on hepatoma cancer cells and it must be used with caution.


This research was supported by grants from the Department of Pathobiology, Faculty of Veterinary Medicine, University of Tabriz-Iran.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

 > References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]

  [Table 1]


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