Original article
The new esters derivatives of betulin and betulinic acid in epidermoid squamous carcinoma treatment – In vitro studies

https://doi.org/10.1016/j.biopha.2015.04.003Get rights and content

Abstract

Background

Betulinic acid and betulin are triterpenes with documented cytotoxic properties toward various cell lines. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications.

Objective

To investigate the specific anticancer role of the newly synthesized betulin derivatives in human epidermoid carcinoma cells.

Methods

In the present study we synthesized five amino acid esters of betulin. For the synthesis we selected alanine (Boc-l-Ala-OH, negative control) and four basic amino acids – natural lysine (Boc-l-Lys(Boc)-OH) and three its unnatural derivatives (Boc-l-Dap(Boc)-OH, Boc-l-Dab(Boc)-OH, and Boc-l-Orn(Boc)-OH). Boc-protected amino acids were most convenient for the synthesis. All new esters have one (betulin-l-Ala-NH2) or two free amino groups which significantly increase their solubility in water and facilitate their transport through the cell membrane. It is worth noting that the biological activity of new esters of betulin is positive correlated with the length of the side chain of l-amino acid. The highest biological activity displayed compound containing lysine side chain (Lys, –CH2–CH2–CH2–CH2–NH2). Considering the biological activity, other derivatives can be set in the following series: Orn (–CH2–CH2–CH2–NH2) > Dab (–CH2–CH2–NH2) > Dap (–CH2–NH2) > Ala (CH3) > betulin. New betulin esters were tested in normal human keratinocytes (HaCaT) and human epidermoid carcinoma cells (A431). To assess cytotoxicity, MTT test was performed after 24, 48 and 72 h of incubation with the test compounds at a concentration range of 0.75–100 μM. In case of apoptotic activity, a TUNEL method and comet assay were performed. Additionally expression of caspase-3 and PARP1 was evaluated immunocytochemically.

Results

The highest cytotoxicity in cells induced skin cancer new compounds, particularly compound containing a lysine side chain (IC50 = 7 μM) and ornithine (IC50 = 10 μM). The highest number of apoptotic cells was observed in case incubation with compound containing Orn, Dab and Dap side chain.

Conclusions

The new betulin ester derivatives display enhanced antitumor activity compared to their non-modified precursors. It is worth emphasizing their specific toxicity against epidermoid carcinoma cells.

Introduction

The new anticancer agents and therapies are constantly searched. Researchers focus mainly on new applications of well-known drugs and also look for new, more efficient compounds with anti-tumor activity. Therefore, the development of novel and natural potent, non-toxic anticancer agents is worth a continuous effort. Natural substances derived mostly from plants provide an excellent platform for new drugs discovery. Herbal medicine based on drugs derived from plant in medicinal practice often becomes a kind of an alternative treatment [1], [2]. The majority of anticancer factor are of natural origin. The natural environment is a rich source of such materials which are currently used in oncology either in their intact/raw form, or as chemically modified derivatives (vinblastine, vincristine, paclitaxel, etoposide, teniposide) [3]. Moreover, it is known that natural products have been widely used for fighting human ailments [4], [5]. Recently, the researchers are interested in pentacyclic triterpens. These natural agents are formed by arrangement of squalene epoxide and are found in most plants. One of such compounds with promising anticancer activity are betulin (found mostly in the bark of the birch), betulinic acid (oxidation product of betulin) and their chemical derivatives [6], [7]. Previously reported in vitro and in vivo studies have demonstrated that these compounds have anti-tumor properties, and induce apoptosis in various tumor cells (human melanoma lines Mel-1, Mel-2, squamous carcinoma line A-431, cancer of the stomach sensitive and resistant to daunorubicin) [8], [9], [10], [11], [12].

Betulinic acid and betulin are triterpenes with documented cytotoxic properties against various cell lines [13]. Unfortunately both betulinic acid and its metabolic precursor, betulin, are very poorly soluble in aqueous buffers, thus their bioavailability and bio-distribution are insufficient in terms of medical applications. The newly synthesized betulin derivatives have a much better solubility in water while maintaining a high biological activity [11], [14]. In 2006, the action of triterpenes derived from bark of the birch on tumor squamous cell line A431 and also on normal human keratinocytes was described. The preliminary studies were performed in clinical trials in patients with a clinical diagnosis of actinic keratosis using the suspended in oil triterpenes extract [15]. Moreover, these compounds can induce apoptosis in cancer cells derived from keratinocytes. It was also demonstrated that betulin derivatives can stimulate differentiation in normal keratinocytes [16], [17]. Actinic keratosis (AK) is in the skin considered as precancerous lesion. These lesions are occurred in the skin damaged by the sun's rays. From the focal lesion can develop squamous cell carcinoma. The malignant neoplasm derived from keratinocytes can metastasize. Despite the raised frequency of skin cancer, the knowledge is still very poor and requires further research. The problem is very complex due to the trends in fashion for sunbathing, continues exposition on ultraviolet radiation, ozone depletion expanding and lengthening the survival of the population. Currently there are many applied treatments of AK, however none of them is sufficiently effective. The local formulations containing cytostatic (5-fluoroacyl) and immunomodulatory substances (imiquimed, diclofenac) are associated with large inflammatory reaction in the treated skin. Moreover, laser therapy and photodynamic therapy are very expensive methods, requiring specialized equipment and proper photosensitizers. Therefore, the new method and chemotherapeutics are needed for AK treatment. Recent studies showed the high efficiency in the AK treatment of the oil extract from triterpenes, derived from the birch bark. Moreover, the obtained results indicated very good skin tolerability [15], [17], [18].

Our research group derivatization of betulin from birch bark with selected natural amino acids (National patent application No PL211589 of 28.12.2011), which had better water solubility than betulin. We have also found, that new compounds have proapoptotic activity in cancer cells [11] what was has been also indicated in other studies [19], [20]. The purpose of our studies was to estimate the anticancer potency of new derivatives of betulin and betulinic acid in malignant cells derived from keratinocytes. The effect of those compounds will be also evaluated on model cells – normal human keratinocytes.

Section snippets

Cell culture

The human epidermal cancer cell line was used (A431), and normal keratinocytes (HaCaT) was use as a control. The epidermoid squamous carcinoma cell line A-431, derived from an 85-year-old female, is one of a series of cell lines established from solid tumors by D.J. Giard et al. [21]. The HaCaT cell line was a well-known immortalized human keratinocyte cell line, which was utilized for their high ability to proliferation and differentiation in vitro (ATCC) The cells were grown in DMEM (medium,

Survival assay

The highest cytotoxicity in cells skin cancer induced new compounds, particularly compound containing a lysine side chain (IC50 = 2.25 μM for 72 incubation hours and 7.28 for 48 h incubation) and ornithine (IC50 = 4.5 μM for 72 h incubation and 10 μM for 48 h incubation) in comparison to betulin and betulinic acid. For betulin-Dab-NH2, betulin-Dap-NH2l and betulin-Ala-NH2 the effect was significant only after 72 h incubation (Table 2). The enhanced antitumor activity was observed in the new betulin esters

Discussion

Multiple anti-precancerous lesion (actinic keratosis), basal skin cancers, derived from keratinocytes, therapies including surgical procedure, chemotherapy, photodynamic therapy have been developed. Some of them can cause side effects like baldness, immunodeficiency, organ damage and are not enough effective in skin cancer treatment [24], [25]. Majority type of cancer cells is resistant to apoptosis. In many cases malignant cells are characterized by mutation in genes responsible for this death

Conclusion

The results obtained in the current study indicate that new derivatives of betulin and betulinic acid can be effectively applied in human squamous cell lines. The most benefit is that the new compounds are safe for normal human keratinocytes. These findings demonstrate that new modified compounds derived from betulin have the high therapeutic potential in actinic keratosis.

Disclosure of interest

The authors declare that they have no conflicts of interest concerning this article.

Acknowledgments

The study was funded by the National Science Centre project number: 0275/B/P01/2010/38 (M. Drag-Zalesinska).

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