Millions of people worldwide are affected by skin diseases, which range from chronic diseases (e.g., atopic dermatitis and psoriasis) to neoplasm. Despite being potentially curable, the latter is linked with a significant mortality rate(1–3). In 2020, the International Agency for Research on Cancer (IARC) released data indicating that there were approximately 1.52 million new cases of skin cancer. Regrettably, this alarming number was accompanied by 121,000 deaths(4, 5). Skin cancer claims a life every four minutes, amounting to one person succumbing to the disease within this brief interval(6). Moreover, the outlook for the next 20 years, according to the agency's projections, is bleak. Melanoma incidence and mortality rates are expected to surge by 78% and 73%, respectively (4, 5), casting a shadow over the future landscape of skin cancer (4, 5).
Various skin layers can undergo neoplastic changes, with the epidermal layer being one of them. Epidermal cell malignancies can be classified into two types: melanoma and non-melanoma skin cancers. The origin of these cancers is determined by whether the cells are melanocytes or keratinocytes and Merkel cells, respectively(7, 8). Indeed, the uncontrolled proliferation and malignant transformation of melanocytes give rise to an aggressive disease known as melanoma(9, 10). Melanocytes are derived from a structure known as the neural crest in vertebrate embryos and they play a crucial role in the production of melanin (melanogenesis) and storing it until it is transferred to keratinocytes(11–13). In fact, melanin, a vital pigment, is accountable for the color of the skin, hair, and eyes. Furthermore, the protection of the skin against sun exposure, the elimination of reactive oxygen species (ROS), and the storage of ions are all important tasks carried out by it(14). As mentioned earlier, melanocytes are mainly located in the stratum Basale (the innermost layer out of four layers of the epidermis)(15). Likewise, cutaneous melanoma arises from the malignant transformation of epidermal melanocytes, regardless of whether it has the ability to metastasize or not (16). Based on studies, the majority of diagnosed cases consist of cutaneous melanoma(17, 18). In addition, melanocytes are not limited to the skin and can be found in other tissues as well. These tissues include hair follicle bulbs, eyes, inner ear, mucosa, and central nervous system. In these cases, melanocytes are present in a non-cutaneous form(19, 20).
Melanoma, despite being the least prevalent type of skin cancer, is highly aggressive and lethal in nature(10, 21, 22) and the outlook for the coming years regarding its incidence and mortality will be accompanied by an increasing trend, with up to 132,000 diagnoses annually(23, 24). IARC's estimates indicate that there were roughly 325,000 new cases of cutaneous melanoma in 2020(25). moreover, it predicted that by 2040 melanoma new cases and related deaths increase by approximately 57 and 68%, respectively(26, 27). Melanoma, accounting for 1.7% of all cancer diagnoses, is considered to be among the most prevalent forms of cancer worldwide and probably has been leading to 57,000 fatalities in the same period(28). That is, not only is melanoma known as the third most common malignancy in the world but also, according to the GLOBOCAN reports, it is responsible for 324,635 new cancer cases and 57,043 deaths globally in 2020(28, 29). Unfortunately, the annual incidence rates of melanoma worldwide are increasing rapidly every year, with a more significant increase than any other major cancer type(30).
The conversion of melanocytes into malignant cells is an intricate procedure that arises from the interplay of various factors that can be modified or non-modifiable(17). The most influential modifiable risk factor of melanoma is Exposure to UV radiation from sunlight or the use of tanning devices which is responsible for around 60–70% of all diagnosed cases. Besides, ultraviolet radiation escapes melanin absorption, Consequently, genetic changes occur due to as a result of two actions: (i) UVA-generated reactive oxygen species (ROS)and/or (ii) direct UVB-driven DNA mutations/breaks. (31–37). Other risk factors include medications and environmental exposure to chemicals, age, sex, ethnicity, individual phenotypic characteristics (e.g., skin and light eyes, red or blond hair, and high density of freckles), clinical characteristics of the patient(e.g., increased number of common nevi or presence of atypical nevi), history of blistering sunburns at a young age, personal and family history of skin cancers, and personal history of diseases that compromise the immune system, genetic alterations and specific genetic conditions (e.g., Albinism or Xeroderma pigmentosum), which can contribute to a change in the molecular machinery of melanocytes and eventually lead to the development of tumors(10, 17, 38–41).
Numerous mutated driver genes crucial for the development and carcinogenesis of melanoma have been identified. These genes are concentrated in various signaling pathways essential to the melanoma's progression, including the mitogen-activated protein kinase (MAPK) pathway, phosphoinositide 3-kinase (PI3K), tumor suppressor retinoblastoma, cell-cycle regulation pathway, pigmentation-related pathway, p53 pathway, epigenetic factors, and several other pathways (42, 43) This article specifically focuses on the MAPK pathway.
Among the various intracellular signaling pathways, the MAPK pathway holds a more prominent role in cell proliferation, differentiation, apoptosis, angiogenesis, and tumor metastasis (44). Furthermore, this complex interconnected signaling cascade is frequently triggered to promote the rapid growth of tumor cells and involvement in oncogenesis, tumor progression, and drug resistance(42, 45). Upon the extracellular binding of growth factors to receptor tyrosine kinases (RTKs), a cascade of intracellular events, leads to the sequential activation of Ras, Raf, MEK, and ERK that is responsible for the regulation of numerous oncogenic biological activities. On the other hand, among various cancer types, melanoma has the highest rates of somatic mutations(46). The hyper-activation of the MAPK pathway in melanoma is primarily driven by mutations in critical signal components, including BRAF, NRAS, NF1, and KIT(42). Genetically, BRAF-activating mutations are prevalent in over 50% of melanoma cases, and the most frequent mutation (over 90%) involves a substitution of Valine (V) with Glutamic acid (E) at second position of codon 600 (p.V600E) and contribute to constitutive signals in the MAPK pathway(47–50). In fact, BRAF—located on chromosome 7q34— is a serine/threonine protein kinase that plays a pivotal role in activating the MAP kinase/ERK-signaling pathway. It is worth noting that BRAF is the family member that Ras most easily activates it(51, 52). As a result, the BRAFV600E mutation has been considered a promising therapeutic target for melanoma, leading to the emergence of dedicated inhibitors tailored to combat BRAFV600E(53). The BRAFV600E mutation, the most frequently occurring mutation associated with intermittent sun exposure, entails a valine-to-glutamate point mutation at residue 600. This mutation results in the hyperactivation of the mitogen-activated protein kinase (MAPK) pathway, playing a pivotal role in cell proliferation (54, 55).
Surgery, radiotherapy, chemotherapy, immunotherapy, and targeted therapy are the currently existing treatment options for melanoma(23, 56). The initial course of action for melanoma treatment is typically the surgical removal of the affected area, ensuring adequate margins(57). Nevertheless, especially in some patients at stages II and patients at stages III and IV (or when a patient is diagnosed with distal metastasis or inoperable melanoma), surgery alone has limited curative potential. As a result, adjuvant treatments such as radiotherapy, chemotherapy, immunotherapy, or targeted therapy are often employed(58–61).
Chemotherapy is currently one of the most widely utilized therapeutic methods in the field of cancer treatment(62, 63). Furthermore, it maintains its status as a vital tool in the management of melanoma. Despite the absence of conclusive evidence supporting an enhancement in overall survival, chemotherapy remains a significant and promising treatment avenue, particularly for palliative care in patients with melanoma (64).
To the best of our knowledge and based on the assessment of multiple chemotherapeutics for treating advanced melanoma, it is noteworthy that the DNA alkylating agent dacarbazine (DTIC) stands as the sole drug approved for use by both the FDA and EMA (64, 65). Chemotherapy is often necessary for advanced melanomas. For more than four decades, Dacarbazine (DTIC), also known as 5-(3,3-dimethyltriazeno)imidazole-4 carboxamide, has been the standard chemotherapy drug employed in first-line melanoma treatment, despite less than satisfactory results (66–68). DTIC, a methylating agent, induces DNA damage resulting in cell cycle arrest, and apoptosis(69). The active compound 5-(3-methyl-1-triazeno)imidazole-4-carboxamide (MTIC) is formed through the conversion of DTIC, a pro-drug, by cytochrome P450 isoforms in the liver.(70) The half-life of MTIC is extremely short, causing it to spontaneously decompose into two substances: 5-aminoimidazole-4-carboxamide (AIC), an inactive derivative involved in purine de novo synthesis, and methyl diazonium cation, an alkylating agent(71). Exposure to light can also trigger the activation of DTIC, owing to its light-sensitive and unstable nature. This activation process results in the production of two compounds, namely 5-diazo imidazole-4 carboxamide and 2-azaipoxantine which have shown cytotoxic effects in vitro but not in vivo(72, 73). In contrast, clinical trials have demonstrated that this medication exhibits a moderate level of effectiveness in treating tumors. Nonetheless, dacarbazine remains a prominent therapeutic option in the field of chemotherapy for patients with metastatic melanoma(74).The response rate of DTIC in several clinical trials was generally observed to be around 10 to 20%, with the majority of responses being partial and not enduring over time. Additionally, the prevalent adverse side effects consist of severe nausea and vomiting, cardiac and hepatic toxicity, myelosuppression, and mucocutaneous toxicity(66, 75–77).
Furthermore, apart from DTIC, several other chemotherapeutic agents have been utilized off-label, despite lacking official approval. Notably, these agents include temozolomide (TMZ), nitrosoureas, paclitaxel, docetaxel, and cis/carboplatin(65, 78). Although Temozolomide (TMZ), the dacarbazine analog, initially approved for glioblastoma, is frequently employed in the treatment of metastatic melanoma(66). On the other hand, Temozolomide, an alkylating cytostatic medication, is being increasingly utilized in the management of melanoma, anaplastic astrocytoma, and glioblastoma multiforme(79). Unlike Dacarbazine, TMZ spontaneously hydrolyzes in an aqueous solution to form MTIC and exhibits activity in vitro(80, 81). Indeed, Temozolomide, similar to Dacarbazine, functions as an alkylating agent that causes DNA damage by introducing alkyl groups to guanine bases. Consequently, it leads to cell death(82, 83). The efficacy of both drugs, TMZ and DTIC, was observed to be similar in a phase III clinical trial involving 305 volunteers with advanced disease. The objective response rate for Temozolomide (TMZ) and Dacarbazine (DTIC) was reported as 13.5% and 12.1%, respectively (84). Furthermore, the advantages of TMZ include its oral administration and its wide tissue distribution, facilitated by its ability to traverse the blood-brain barrier—a crucial benefit in the context of managing brain metastases (66, 84–86).
Nevertheless, conventional chemotherapy is burdened with numerous drawbacks including inadequate solubility, and excessive utilization of surfactants to enhance drug solubilization, thereby causing systemic toxicities and adverse side effects(63). Furthermore, the presence of insufficient drug concentration within tumors contributes to the development of a population of drug-resistant cancer cells(87). Chemotherapeutic drugs not only harm healthy cells but also induce the emergence of multidrug resistance (MDR), ultimately leading to unfavorable therapeutic outcomes(88). Besides, the lack of selectivity in most therapeutic agents can cause considerable damage to healthy cells. The low permeability of these agents restricts their ability to penetrate the tumorous tissue, thereby requiring higher doses and more frequent administration of these agents(89). In other words, the major disadvantages of chemotherapy include its non-specificity towards tumor cells and the subsequent inadequate accumulation of drugs within the tumor microenvironment. Therefore, the therapeutic gains are confined, while the occurrence of unfavorable effects is significant(66, 90, 91).
While chemotherapy remains a viable treatment option in the management of melanoma, especially in instances of palliative care or disease relapse, new therapeutic choices are given preference in the advanced stages of metastatic melanoma (66).
lately, RNA molecules have been receiving significant attention in the field of cancer therapeutics development(92). RNA interference (RNAi) is an endogenous post-transcriptional regulatory mechanism that involves the specific silencing of genes through sequence recognition. This intricate process relies on non-coding RNAs such as microRNAs (miRNAs), small interfering RNAs (siRNAs), long non-coding RNAs (lnc-RNAs), and circular RNAs (circ-RNAs) that in a sequence-specific manner, messenger RNAs (mRNAs) are targeted and silenced(93). The application of small interfering RNA (siRNA) derived from RNA interference (RNAi) technology is increasingly being recognized as a powerful approach in the management of cancer and shows promise as a therapeutic strategy(94, 95). RNA (siRNA) can effectively disrupt cellular pathways By efficient suppressing genes, thus offering potential advancements in therapeutics for diseases arising from abnormal gene expression(94).
siRNA, a distinctive class of duplex RNAs measuring 20 to 24 nucleotides in length, undergoes identification and cleavage by the enzyme DICER—a member of the RNAse III family. Functionally, siRNAs induce gene-specific cleavage by binding to mRNA through complementary base pairing (96, 97). Short interfering RNAs (siRNAs) are duplexes processed from long double-stranded RNA (dsRNA). They comprise a sense strand (passenger strand) and an antisense strand (guide strand). The siRNA molecule is loaded onto an RNA-induced silencing complex (RISC), where it interacts with its Argonaute 2 component. This interaction leads to the unwinding of the duplex and subsequent degradation of the passenger strand. Ultimately, the antisense strand, which matches the target mRNA, directs the RISC complex towards the mRNA(97–100). Cleavage of mRNA occurs as a result of the catalytic function of RISC protein (Ago2), a member of the argonaute family.(101)
It is worth noting that the intricate signaling pathways in cancer cells are associated with various mechanisms that allow them to evade programmed cell death. Consequently, developing effective treatments for cancer encounters significant challenges (102). Studies reveal that metastatic melanoma, being one of the most heterogeneous human cancers, demonstrates a high level of biological complexity throughout disease progression (103, 104). Given this complexity, the application of combination therapy has been extensively researched and proven to be a successful approach in treating various types of cancer (105).
Combination therapy encompasses the integration of two or more therapeutic agents and is considered a cornerstone in the field of cancer therapy. The amalgamation of anti-cancer medications results in a more effective treatment approach than using monotherapy, as it targets critical pathways in a synergistic or additive way (106).
Combination therapy encompasses the use of multiple chemotherapeutic drugs and the integration of chemotherapeutic agents with various treatment modalities such as surgery or radiation (62, 105). As previously highlighted, compared to monotherapy, combination cancer therapy offers several advantages. These include enhancing efficacy through synergistic effects, such as reducing tumor growth and metastatic potential, arresting mitotically active cells, diminishing cancer stem cell populations, inducing apoptosis, and overcoming drug resistance (62, 106).
Recently, the combination of chemotherapeutic drugs and siRNAs has provided a novel perspective on the targeted management of cancer, captivating the attention of researchers worldwide. Utilizing a dual approach of siRNA-based oncogene interference and chemotherapeutic drug-induced tumor cell destruction not only reverses chemotherapeutic drug resistance and efficiently decreases unwanted damage to healthy cells but also exhibits a synergistic and combinatorial anticancer impact(107, 108). Likewise, this strategy is known as a valuable and safe approach to the treatment of MDR tumors(109). Combining siRNA with chemotherapeutic drugs offers a highly advantageous strategy as it allows for the manipulation of various mechanisms and regulatory proteins linked to tumor cell growth, development, metastasis, and drug resistance. This strategy facilitates the attainment of heightened therapeutic effectiveness by concurrently targeting multiple factors (110). The utilization of drug/gene co-delivery is emerging as a valuable method for precisely addressing cells at the molecular level. Thus, within this framework, the integration of siRNA-chemotherapy emerges as a pivotal element in the formulation of a comprehensive combination therapy (62).
In recent years, substantial efforts have been invested in the development of innovative therapeutic strategies to combat melanoma (111). Despite these endeavors, the aggressive and heterogeneous characteristics of melanoma have led to a scarcity of treatment alternatives (112). Although immune and targeted therapies have enhanced the life expectancy of melanoma patients, instances still exist where patients encounter relapse or exhibit resistance to these treatment regimens (16). Consequently, the development of innovative and enhanced treatments to identify a wider range of effective therapeutic options continues to be a priority for researchers. This commitment is underscored by the substantial number of ongoing preclinical and clinical trials, affirming the dedication to advancing melanoma treatment strategies (16, 113).
In effect, the employment of RNA interference (RNAi) as a propitious strategy for treating cancer is gaining momentum and the co-delivery strategies of siRNA and chemotherapeutic drugs have demonstrated remarkable antitumor effects in the management of various types of cancers(62). Thus, the aim of the present study is to investigate the therapeutic effects of conventional chemotherapy drugs such as Dacarbazine and Temozolomide, both individually and in combination with BRAF(V600E) siRNA, and evaluate the probability of their synergistic effects on the A375 human melanoma cell line for more effective treatment.