Mumio (Shilajit) as a potential chemotherapeutic for the urinary bladder cancer treatment

Mumio (Shilajit) is a traditional medicinal drug known and used for hundreds of years. Bladder cancer is one of the most common cancer types and better treatments are needed. This study analysed the in vitro effect of Mumio on urinary bladder cancer cells (T24 and 5637) in comparison to normal uroepithelial cells (SV-HUC1). Cytotoxicity of Mumio was analysed in these cell lines via MTT and real-time cell growth assays as well via the assessment of the cytoskeleton, apoptosis, and cell cycle. Mumio affected the viability of both cell types in a time and concentration dependent manner. We observed a selectivity of Mumio against cancer cells. Cell cycle and apoptosis analysis showed that Mumio inhibited G0/G1 or S phase cell cycle, which in turn induced apoptosis. Our results showed that Mumio was significantly more cytotoxic to urinary bladder cancer cells than to normal cells. These results are promising and indicate Mumio as a great candidate for urinary bladder cancer treatment and further investigations should be performed.

. These studies found that the main components of Mumio are humus (60-80%), benzoic acid, fatty acids, ichthyol, ellagic acid, resin, triterpenes, sterol, aromatic carboxylic acids, bioactive 3,4-benzokoumarins, amino acids, phenolic lipids and microelements. The most important role among these have the following bioactive molecules: dibenzo-alpha-pyrones, humic acid and fulvic acid. These components mediate Mumio's strong antioxidant activity and are carriers for active ingredients. The studies carried out by Ghosal et al. showed that these acids inhibit lipid peroxidation and possess the ability to recycle ascorbic acid and thereby exhibiting significant antioxidant activity 7,11,[13][14][15] . Mumio is also a rich source of amino acids, especially exogenous, such as methionine, leucine and threonine, and also endogenous such as histidine, proline, glycine, tyrosine, arginine and aspartic acid 9,10 .
Cancer is one of the most common diseases in this day and age. Despite the development of new and improved treatment and diagnosis methods, it is still the second most common cause of deaths. In 2018, urinary bladder cancer was the sixth most common cancer among men, and seventeenth among women 16 . About 90% of patients are over 55 years old. The treatment methods depend on the cancer stage in bladder cancer. The most commonly used methods are surgical: transurethral resection of bladder tumour (TURBT) for non-invasive tumours and radical cystectomy for invasive tumours. Both of these surgical procedures are usually combined with either chemotherapy, radiotherapy or immunotherapy, to increase the probability for complete depletion of the carcinoma cells 17 .
Based upon the aforementioned properties of Mumio, we hypothesized that administration of this substance may improve the effectiveness of bladder cancer therapy or that it can be used as an adjuvant to support current therapies. Thus this study investigated the effects of Mumio on urinary bladder cancer cells (T24 and 5637) in comparison to normal uroepithelial cells (SV-HUC-1, Fig. 1).

Results
Cell morphology. We did not observe any changes in cell morphology after 24 h incubation in SV-HUC-1 cells. We only observed a lower number of attached cells at the highest concentrations of Mumio. After 48 h incubation 200 μg/ml of Mumio affected the cells, an effect that increased with increasing drug concentrations. Cell shrinkage, a lower cell number and rounding were observed at 500 μg/ml. After 72 h of drug exposure, cells were affected at 200 μg/ml (Fig. 2). Cell number and morphology was affected at 200 μg/ml in T24 cells at all studied time points. Cell shrinkage, rounding and a lower cell number were observed with increasing concentrations of Mumio (Fig. 3). An increased number of round, detached cells was observed with 500 μg/ml and higher concentrations in 5637 cells. A similar trend was noted after 48 h with a striking difference at 500 and 1000 μg/ ml and after 72 h fewer cells were present at 200 μg/ml. At 1000 μg/ml after 72 h incubation most of the 5637 cells lost their normal morphology, and a considerable amount of cell debris was observed (Fig. 4).
Cell viability and selectivity index. Mumio was cytotoxic in T24 cells in a time and concentration dependent manner. There were no significant differences in cells viability after 24 h at 200, 500 and 800 μg/ml Mumio in SV-HUC-1 cells. However, cell viability at all tested concentrations was lower compared to control. A significant difference was also noticed at 1000 μg/ml. Comparison between SV-HUC-1 and cancer T24 cell line after Mumio treatment showed that normal cells at all tested concentrations after 48 and 72 h were significantly lesser effected. After 24 h of treatment a significant difference between SV-HUC-1 and T24 cells was only noticed at 800 μg/ml Mumio. 5637 cancer cells were significantly more viable than SV-HUC-1 at all tested Mumio concentrations after 24 h. The cancer cells were significantly lesser affected at 200 and 500 μg/ml Mumio after 48 h of incubation, while the opposite was observed at 1000 μg/ml. A similar ratio as seen after 48 h at 200 and 500 μg/ ml was observed after 72 h (Fig. 5).
MTT assay enabled the calculation of the lethal concentration (LC). The obtained values were used in further analysis ( Table 1) Real-time cell growth analysis. The results obtained with xCELLigence RTCA DP system confirmed that the lethal concentrations acquired via the MTT assay caused the decrease of viable cells by 10, 50 and 90%. Results were confirmed for both cell lines for all three incubation times (Fig. 6).
F-actin staining. Staining of F-actin microfilaments revealed that Mumio at LC 10 in SV-HUC-1 cells did not cause any significant reorganization of the actin filaments. However, the LC 50 concentration caused degradation of stress actin fibres indicated by increased round aggregates of actin, while at LC 90 almost no stress fibres were observed. A similar trend was observed in T24 cells, with no effect at LC 10 , noticeable smaller stress fibre at LC 50 , and round actin aggregates instead of fibres at LC 90 . However, the cell shape was preserved. Incubation at LC 10 caused a noticeable lower number of long stress actin fibres in 5637 cells. After exposure to LC 50 , the stress fibres had a less organized structure and more short fibres were present. Exposure with Mumio at LC 90 completely changed the cell morphology and only round actin aggregates were observed (Fig. 7). Apoptosis. Analysis of apoptosis revealed increased late apoptotic cells in SV-HUC-1 treatment group after exposure to LC 90 with a significant decrease in viable cells (p < 0.01) when compared to control. No effect was observed at lower concentrations. A significant increase in apoptotic cells was noticed after Mumio exposure at LC 50 (p < 0.05) and LC 90 (p < 0.01) in T24 cells. This occurred along with a significant decrease viable cells. 5637 cells treated with LC 90 had significantly fewer viable cells (p < 0.001) along with a significant increase in late apoptotic cells (p < 0.001). There were no significant changes at the lower concentrations (Fig. 8).

Discussion
Numerous studies have confirmed the widespread use of Mumio in traditional medicine mainly because of its anti-ulcerogenic, anti-inflammatory, antioxidant, immunomodulatory, memory enhancement and anxiolytic activities 8,11 . A clinical randomized double-blind placebo-controlled trial conducted by Sadeghi et al. found a beneficial effect of oral consumption of 500 mg Mumio capsules in the healing of Tibia fracture. The time to full recovery was significantly shorter and no increased adverse effects 18 . This study indicates that the oral consumption of 500 mg of Mumio does not cause adverse effects, and can be considered for application to other diseases. www.nature.com/scientificreports/ Mumio is an adaptogen used for urinary tract problems, sexual dysfunctions and improved prostate health. A study in 1997 showed that Mumio has a beneficial effect on bladder obstruction in patients with benign prostate hypertrophy 6,11,19 . Mumio has antimicrobial activity against various strains of pyogenic microorganisms, which is associated with the presence of benzoic and fulvic acid. These antibacterial properties of Mumio can be supportive for bladder cancer treatment and prevention according to the two-hit hypothesis as it not only impairs cancer cell growth but also prevents chronic inflammation, which are considered to be important factors in initiation and progression of bladder cancer 20 . Ellagic and tannic acids are natural polyphenolic antioxidants that inhibit superoxide anion radical production and phospholipase A2 activity. Khanduja et al. and Thresiamma et al. showed that these acids have anticarcinogenic and radioprotective properties 21,22 . In 1991, Ghosal et al. found that Mumio extract significantly inhibits cancer cell proliferation in Ehrlich ascites 4 . Moreover, safety of long-term administration of Mumio was evaluated in a rat model by Velmurugan et al. They showed that daily Mumio administration for 91 days caused only minor changes in the liver of those animals that received the highest concentration of Mumio. These changes correlated with an excess amount of iron in the liver showing potential mechanism of liver changes development 9 .
To date only a few studies evaluated effect and the mechanisms of action of Mumio on cancer cells. Pant et al. showed that Mumio inhibits growth of hepatocellular Huh-7 cancer cell line and induces apoptosis in vitro 23 . In our study a significant increase in apoptotic cells was also observed after incubation of T24 cancer cells with Mumio at its LC 50 and LC 90 . Additionally, late apoptotic cells were also increased after exposure to Mumio at its LC 90 in 5637 cancer cells. In SV-HUC1, normal uroepithelial cells, increased late apoptotic cells were only We also evaluated different cell types, and found similar effects. We found stronger cytotoxicity in one of the tested cancer cell lines when compared to normal cells of the same organ. A direct comparison to our results is not possible because we used lower concentrations of Mumio (25-200 μg/ml). Jafari et al. assessed the effect of Mumio on breast cancer cells, MCF-7, and lung carcinoma cell lines, A549. They found a concentration dependent cytotoxic effect of Mumio on both cancer cell lines. Their results in the A549 cells were similar to those found by Thawatchai et al. 25 . In contrast to these studies, our study assessed the effect of Mumio between normal and cancer cells of the same organ. This approach enables a better assessment of the drug effectivity and safety at the initial research stage. All LC values obtained in this study were higher for normal cells than for T24 cancer cells. Moreover, the calculated selectivity index for T24 after 48 and 72 h exposures were higher than 2.0 indicating a Mumio also affected the cytoskeleton of these two selected cancer cell lines. The actin filaments were damaged but spread evenly while the cell shape was preserved at the LC 90 concentration in T24 cells. However, the 5637 cells rounded up and the actin filaments were concentrated as round aggregates. Additionally, Mumio affected the cell cycle differently in these two cancer cell lines such that the cell cycle was arrested in different phases. T24 cells were arrested in G0/G1 cell cycle while in 5637 cells were inhibited in S phase. This phenomenon may be explained by the multicomponent composition of Mumio as different components can affect cells with a different metabolism and proliferation rates. This suggests that potential long-term administration of Mumio may be beneficial in the prevention of development or relapse of urinary bladder cancer. Our research showed that Mumio at the tested concentrations has a stronger effect on one of the bladder cancer cell lines than on normal uroepithelial cells. A favourable trend of Mumio was observed in the second tested cancer cell line with prolonged exposure indicating potential long term administration benefits. These promising results form the basis for further investigations of potential applications of Mumio in the treatment and prevention of bladder cancer (Fig. 1).

Conclusion
Our results indicate that more interest should be given to the evaluation of Mumio's potential use for cancer treatment. In can be potentially difficult to analyse the distribution of Mumio to various tissues because of the complex composition. Due to these considerations, we suggest to further investigate intravesical application together with long term oral administration of Mumio for bladder cancer treatment. Moreover, long term administration of Mumio was found to be save in an animal model and clinical studies did not show significant adverse events after Mumio administration 9,18 . However, it is important to consider the depletion of insoluble mineral salts in order to limit the risk of bladder stones and irritation of bladder wall, which could create a niche for implantation of tumour derived cancer cells.      Statistical analysis. Each experiment was performed at least in triplicate. Mean cell viability was expressed as a percentage relative to the control. All data are presented as means ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA with Tukey post-hoc (for cell viability) or two-way ANOVA