Antiproliferative activity in 2D and 3D melanoma models of extract from Buddleja saligna willd and isolated compounds

Abstract

Cancer is one of the most lethal diseases worldwide, with millions of people being diagnosed each year, it is estimated that 1 in 6 deaths can be attributed to this disease. In 2020 approximately 10 million deaths occurred globally due to cancer (Ferlay et al., 2020). The World Health Organisation (WHO) reported that in 2018 there were 107,467 new incidences and 57,373 deaths of cancer cases in South Africa (World Health Organization, 2019). It is forecasted that the incidence rate during the period of 2019-2030 will increase from approximately 62 000 to 121 000 (Finestone, 2022). Melanoma is a malignant neoplasm that develops from melanocytes which are present in pigmented tissue found in the body (Hurt, 2008). Cutaneous melanoma accounts for approximately 90% of melanoma cases and due to its high metastatic potential, it is the most lethal type of skin cancer (Erdei and Torres, 2010). The estimated survival rate for patients with early-stage melanoma is approximately 99%, however melanoma that has metastasised to other organs has a significantly decreased survival rate (Erdei and Torres, 2010). Advanced stages of melanoma become more complex to treat, often resulting in a poor prognosis (Matthews et al., 2017), stage 4 metastatic melanoma has an average ten-year survival rate of less than 10% (Balch et al., 2001). Conventional 2D cell culture techniques have been used for many years as a way to discover potential drug candidates that can be considered for further pre-clinical studies. However, these methods fail to represent the complexities of the in vivo environment. This leads to drug candidates being unsuccessful in later stages of pre-clinical studies as well as large costs involved to establish the drug. It is estimated that it can take 12-15 years and up to $2.8 billion to successfully develop 1 drug (Singh et al., 2023). During the first three phases of clinical trials approximately 90% of the drug candidates will fail (Sun et al., 2022). Although 2D methods have served as workhorses for decades, recent years have witnessed a surge in awareness regarding the significance of the cellular microenvironment. This shift has prompted the exploration of innovative solutions in cell culture research. Cellular behaviour such as differentiation, migration, growth, and mechanics are profoundly influenced by the microenvironment surrounding it as well as the elements present biochemically. Understanding these interactions may offer some insight into in vivo processes. In order to bridge the gap between traditional 2D and in vivo tissue environments, 3D cell culture methods were developed. These techniques are improving constantly and although it can be a challenge, this is the way forward with regards to the investigation of drug candidates’ anticancer activities. Buddleja saligna is known as the false olive (English), umBatacwepe (Siswati), unGqepe (Zulu) and Lelothwane (South Sotho). It is an evergreen tree, which is distributed throughout South Africa and Zimbabwe. Parts of the plant such as the leaves and root is used for different purposes in traditional medicine, including to treat a cough or as an emetic. Apart from the popularity this Buddleja species has gained in traditional medicine, it is also known to have various isolated compounds that have reported biological activity. These activities include antiplasmodial, antimycobacterial, antimutagenic and antioxidant properties. These compounds include α-spinasterol, betulonic acid, betulone, oleanolic acid and ursolic acid. A previous study reported the efficacy of the ethanolic extract of B. saligna against human malignant melanoma cells (UCT-MEL-1) and human keratinocytes (HaCaT), with exhibiting IC50 values of 31.80 and 58.65 μg/ml against the two cell lines respectively. The extract of the plant and a semi-pure fraction isolated from the leaves and stems containing oleanolic and ursolic acid indicated potent antiproliferative effects and exhibited anti-angiogenic potential, indicated by the inhibition of key factors including nitric oxide, cyclooxygenase-2, interleukin-6, interleukin-8, sphingosine kinase 1, and VEGF. The antiangiogenic activity was validated through ex-ovo chorioallantoic membrane assay, substantiating its ability to inhibit the growth of vessel formation. These findings underscore the therapeutic potential of B. saligna as a source of bioactive compounds for combatting cutaneous melanoma. In this study the purified compounds - oleanoic acid and ursolic acids from B. saligna were tested for cytotoxicity in 2D and 3D cell culture models. It was reported that the cytotoxicity against 2D cell culture models indicated IC50 values of 69.29 ± 24.78 μg/ml, 74.49 ± 3.06 μg/ml and 15.11 ± 0.23 μg/ml for the ethanolic extract of B. saligna, oleanolic acid and lastly ursolic acid, respectively, for the A375 cell line. While the IC50 values ranged from 85.50 ± 7.47 μg/ml, 84.69 ± 3.53 μg/ml and 7.57 ± 0.61 μg/ml against the RPMI-7951 cell line for B. saligna ethanolic extract, oleanolic acid and ursolic acid, respectively. This was compared to the 3D cell culture model, which was spheroids formed from the RPMI-7951 cells as well as the HaCaT cells. The spheroid results indicated that two compounds exhibited more than 50% inhibition of the RPMI-7951 spheroids after 72 hours and did not exhibit cytotoxicity against the non-cancerous keratinocyte HaCaT spheroids. These results were further substantiated by the images observed using the fluorescent microscope and the live/dead assay. The semi-pure compounds of oleanolic and ursolic acid were also successfully isolated from the ethanolic extract. This was done by completing various chromatography techniques, such as liquid-liquid chromatography and targeted isolation. The isolation of oleanolic and ursolic acid was confirmed by performing High-performance thin layer chromatography (HPTLC). This allowed the visualisation of the chromatograms and subsequently the specific fingerprints of the compounds as well as the fractions isolated from the ethanolic extract of B. saligna. By combining traditional knowledge with modern scientific approaches, this research contributes to the field of natural product-based drug discovery, advocating for the exploration of indigenous plant species for novel cancer therapies.