Breast Cancer: A Global Concern, Diagnostic and Therapeutic Perspectives, Mechanistic Targets in Drug Development

Cancer is a complex multifactorial process, unchecked and abrupt division, and cell growth—conventional chemotherapy, along with radiotherapy, is used to treat breast cancer. Due to reduce efficacy and less survival rate, there is a particular need for the discovery of new active anticancer agents. Natural resources such as terrestrial/marine plants or organisms are a promising source for the generation of new therapeutics with improving efficacy. The screening of natural plant extracts and fractions, isolations of phytochemicals, and mechanistic study of those potential compounds play a remarkable role in the development of new therapeutic drugs with increased efficacy. Cancer is a multistage disease with complex signaling cascades. The initial study of screening whole extracts or fractions and later the isolation of secondary compounds and their mechanism of action study gives a clue of potential therapeutic agents for future drug development. The phytochemicals present in extracts/fractions produce remarkable effects due to synergistically targeting multiple signals. In this review, the molecular targets of extracts/ fractions and isolated compounds highlighted. The therapeutic agent's mechanistic targets in drug development focused involves; i) Induction of Apoptosis, ii) modulating cell cycle arrest, iii) Inhibition or suppression of invasion and metastasis and iv) various other pro-survival signaling pathways. The phytochemicals and their modified analogs identified as future potential candidates for anticancer chemotherapy.

in the milk glands, while ductal carcinoma began in the breast's milk ducts. Medullary breast carcinoma and tubular carcinomas are the subtypes of invasive breast carcinoma. 10 Furthermore, inflammatory breast cancer type is characterized by inflamed breasts with indentation and thick ridges. Only 1% to 2% of all invasive breast cancers and 1% of all breast cancers are inflammatory breast cancer with low survival rates at all stages. However, non-invasive breast cancer cells restricted to ducts only (do not penetrate surrounding tissues) of the breast. Ductal carcinoma in situ and lobular carcinoma in situ are the two forms of non-invasive breast cancer. [10][11][12][13] PD pharmaceutically described by the infiltration of the nipple epidermis by destructive breast epithelial cells. PD of the breast defined as a skin alteration in the nippleareola region. It is less common and generally linked with in-situ or invasive carcinoma. 14 Breast PT, is a rare tumor, and shows different behavior, as it could be benign (non-cancerous) or malignant (cancerous). 15 PT can cause uncommon fibroepithelial lesions to account for around 0.3% to 0.5% of breast tumors diagnosed in women and has an occurrence of about 2.1 per million. 16 Breast cancer usually classified as two types i) estrogen receptor-positive (ER+) and ii) estrogen receptor-negative (ER-) breast cancer. Estrogen receptor-positive cell lines include MCF-7 and T-47D, while MDA-MB-231, MDA-MB-453 and MDA-MB-468 are estrogen negative receptor cell lines. Which, further characterized as luminal A (ER+, PR+, HER2-), luminal B (ER+, PR+, HER2+), HER2-enriched, basal-like, and normal-like based on progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2) status. 17 The MCF-7 and T-47D cell line together with other breast cancer cell type MDA-MB-231, have been studied in above two-third of the total publications in Medline. 18 Risk factors, diagnostic and therapeutic perspective for breast cancer Common risk factors associated, such as age, gender, family history, breast density, radiation exposure, reproductive factors, genetic mutations, and diabetes. 19 Early screening, detection, and diagnosis, significantly affect the occurrence and survival rate of breast cancer. Several diagnostic approaches include mammograms, ultrasound, magnetic resonance imaging, breast selfexamination, positron emission tomography scan, computerized tomography, bone scintigraphy, chest X-ray, and biopsy. 20 However, due to some limitations of these approaches, such as high cost, time consumption, and age restriction, the development of highly sensitive and earlystage diagnostic techniques required. Different biomarkers such as proteomic biomarkers, gene biomarkers, and various imaging techniques are a useful analytical tool for fast and economic early-stage breast cancer diagnosis. 21 Breast cancer conventional treatment approach involves; (i) surgical removal of cancer cells. (ii) use of chemotherapy coupled with hormonal therapy and gene therapy; and (iii) radiation therapy. 22 Surgery is considered as the earliest method and used for most of the solid tumors. 22 The surgical treatment depends on the stage and tumor form; involves removal of the only lump (lumpectomy) or surgical removal of the entire breast (mastectomy). Breast-conserving surgery includes lumpectomy (removal of lump only or a small number of surrounding tissues), wide excision (partial mastectomy), and quadrantectomy (removal of about one-quarter of the breast. 12 Currently, sentinel lymph node dissection has become a well-known suitable technique as it necessitates the excision of very few lymph nodes, causing very few or no side effects. Over the past decade, advances in sentinel lymph node mapping have enhanced the precision of detecting sentinel lymph nodes from 80% to 92%-98% using different combined modalities. 12,22 Chemotherapy is the most conventional therapy available for malignant cancers. 22 In chemotherapy, anticancer drugs, orally or intravenously given to patients, might cause severe side effects due to non-specific killing of cancer cells. However, radiotherapy is a conventional approach used in the treatment of cancer, along with chemotherapy. 22 For the treatment of HER2-neu positive tumors, trastuzumab, in combination with radiotherapy, is needed. 23 Hormonal therapy studied for the treatment of ER+ breast cancer for several decades. The anticancer drug tamoxifen behaves as an antagonist in the breast, causing a delay in the transcription of estrogen-regulated genes and interrupting in the proliferative effects of estrogen in the breast. Similarly, fulvestrant acts as tamoxifen, but it causes degradation of the ER protein and loss of estrogen and progesterone receptor expression. 24 Menopausal hormone therapy usually restrained from breast cancer survivors because of the risk of reoccurrence. Menopausal hormone therapy provides adequate assistance from climacteric symptoms, but few are associated with enhanced risk of stroke and also breast, ovarian, and endometrial cancers. 25 Gene therapies have developed as promising new treatments for breast cancer. Proto-oncogene and tumor suppressor genes have shown accelerated improvement in gene therapy approaches. 12 Various clinical trials are ongoing to deliver p53 to cancer cells. The viral vectors have employed to transfer a breast cancer gene BRCA1, as a mutation in BRCA genes is also responsible for breast cancer cases. Also, the use of antisense strategies in clinical trials considered the most common approach. Adenoviral gene E1A that interferes with the transcription of erbB-2 can use to inhibit the transcription of overexpressed oncogenes in the treatment of ovarian and breast cancer. 12

Plant-derived anti-breast cancer therapeutic agents
Natural products played a remarkable role in the prevention and treatment of cancer and remained a focus of research in drug discovery. 26 Over 3000 plant species reported having anticancer properties. 27 Plant-derived natural products have significant efficacy in cancer treatment due to reduced adverse side effects as compared to conventional chemotherapy. 28 This review article's primary objective was to study the therapeutic potential of natural products in whole plant extracts/fractions or isolated secondary metabolites in breast cancer treatment. The initial stage in drug discovery is to screen the potential extracts and fractions, which gives the clue of the presence of novel phytochemicals. The knowledge provides a better understanding of the presence of various phytochemicals and their synergistic approach, which nowadays played a remarkable role in combination drug therapy. The extracts/fractions screening reduces the cost of isolation of phytochemicals by revealing a potential agent's presence. The isolated secondary metabolites from various natural sources, mechanistic study (in-vitro), give better molecular fundamental knowledge of the future therapeutic agent. Figure 1 describes the preparation of phytochemical compounds isolated from plants and their use in breast cancer therapy.

Anti-breast cancer therapeutic agents and their molecular mechanistic targets
Several crude extracts/fractions possessing potential natural products have tested on a variety of breast cancer cell lines. The potential phytochemicals induced cytotoxicity on breast cancer through several mechanisms. Such as via induction of apoptosis, cell cycle arrest in cancer cells, inhibition of metastatic potential, obstructing the process of angiogenesis, pro-survival signaling, and autophagy activation. Figures 2 and 3 provide detailed information about potential therapeutic approaches of several antibreast cancer plant species and their mechanism of action, respectively.

Induction of apoptosis
Apoptosis, tightly regulated mechanism of cell death as a result of signal cascades involved during healthy development and morphogenesis. 3, 29 The enzymatic proteins caspases are prominent initiators and executioners in the process of apoptosis. Along with caspases, various pro and anti-apoptotic proteins such as Bcl-2 family proteins, tumor suppressor proteins (p53), cytochrome c release from mitochondria, activation of several death receptors involved in the trigger of apoptosis. Besides, various apoptosis proteins (IAPs) play a vital role in the induction and regulation of apoptosis. 30,31 Apoptosis can occur via both the extrinsic pathway (death receptor-mediated pathway) and the intrinsic pathway (mitochondrial-mediated), and these pathways converge at the execution pathway of apoptosis. 3 Death receptors, DR4, DR5 trigger the extrinsic pathway of apoptosis, up-regulation of pro-apoptotic protein (Bax) and down-regulation of an anti-apoptotic member of Bcl-2, which is essential for the activation of the intrinsic pathway of apoptosis. 3,32 Extract of Phaseolus vulgaris (family Fabaceae) induces apoptosis in MCF-7 and MDA-MB-231 via up-regulating pro-apoptotic protein (Bax) and down-regulated anti-apoptotic protein (Bcl-2, Bcl-xL). 33 Similarly, fruit extract of Momordica cochinchinensis causes the induction of apoptosis in breast cancer (MCF-7) cells via the up-regulation of Bax and enhanced caspase 6, 8, and 9 activity. 34 Also, Fragaria ananassa (Strawberry) methanolic extract induced apoptosis by an intrinsic pathway in T-47D by the up-regulation of Bax, Bid, p73, and down-regulation of BCL-xL. 35 The aqueous extract fraction of Oenocarpus bacaba also induced apoptosis in MCF-7 cells by both extrinsic and intrinsic pathways through activation of caspases-6, -8, and -9. 36 Moreover,

Modulating cell cycle arrest
Cell cycle, remarkable role in cellular genomic integrity, and timely progression of cells. 39 Different phases such as (i) G1-phase (gap 1), (ii) S-phase (DNA synthesis), (iii) G2-phase (gap 2), and (iv) M-phase (mitosis) involve in the cell cycle. In S-phase, DNA synthesis and genome replication occur, required for the transmission of genetic information between generations. The M-phase causes segregation of genetic information, sister chromatids, and cell division. G1 is the gap between M and S phase, while G2 is the gap between S and M phase. These intervals (G1 and G2), essential to ensure that each phase is complete before moving to the next phase. 40,41 Activation of cell cycle check-points usually occurs as a response to replication stress and DNA damage. The activation and inactivation of cyclin-dependent kinases and cyclins play a vital role in the cellular progression and cell cycle regulation. 39,41 The methanolic extract prepared from Allium atroviolaceum (family Amaryllidaceae) induces apoptosis by modulating cell cycle arrest in caspasedependent and p53-independent pathway in the breast cancer cell (MCF-7, MDA-MB-231). 42 Similarly, ethanol extract of Ganoderma lucidum chipped fruiting bodies causes cell cycle arrest in MCF-7 cells by up-regulation of p21/Waf1 and down-regulation of cyclin D1. 43 The crude extracts of Echinophora platyloba, Vernonia amygdalina, Morinda Citrifolia induces apoptosis in MCF-7 and MDA-MB-231 cell lines via G0/G1/S phase cell cycle arrest. 12,44,45 The diethyl-ether extract of Artocarpus altilis and hexane and methylene chloride fractions from roots of Salvia chloroleuca induced apoptosis and sub-G1 peak in T-47D and MCF-7 cells respectively. 46

Inhibition of invasion and metastasis suppression
The conventional therapeutic approaches quite challenging, especially in metastasized cancer. The mechanism of metastasis involves invasion, intravasation, and extravasation. The process of invasion characterizes by the spreading of cancer cells to distant sites via the circulatory system. However, extravasation requires the penetration of cancer cells to the endothelium and the basement membrane. At the point of extravasation, cancer cells can grow at secondary focus. 49,50 The matrix metalloproteinases (MMPs), critical proteins involved in metastasis of tumor cells. The inhibition or blocking of MMPs is an essential target in the suppression of metastatic potential. Other than MMPs, metastasis suppressor genes, MKK4 (mitogen-activated protein kinase 4), BRMS1 (breast cancer metastasis suppressor 1) and NM23 (nonmetastatic gene 23) also play a remarkable role in the inhibition of metastasis. 50,51 Similarly, modulation of uPA, uPAR, and TIMP expression also plays a vital role in the suppression of metastasis. 52 The crude extracts of

Pro-survival signaling pathway
Several pro-survival signaling pathways were determining the fate of a cancer cell and mainly transduced by a complex net of signaling molecule cascade. Pro-survival signaling cascades, IP3K-PKB/Akt, and MAPK, activated by several cytokines and growth factors. The nuclear factor-κB (NFκB) plays an essential role in the regulation of inflammation and immune responses. 58 Blocking of these pro-survival signaling pathways has been widely studied, crucial for the treatment of breast cancer. The previous study shows that methanol extract of Ixeris dentata induced apoptosis in T-47D, MCF-7, SK-BR-3, and MDA-MB-231 via inhibiting Akt and NF-κB signaling pathway. 59   phytochemicals, phenolics are the most structurally diverse. 86 Here, we discuss potential phytochemicals as future anti-breast cancer therapeutic agents and drug development.

Phenolics
Phenolic compounds, widely occurring secondary metabolites isolated from plants and are most structurally diverse among all phytochemicals. 87  The vinflunine, a new synthetic vinca alkaloid, is used in treatment for carcinomas and other malignancies. 99 The vinca alkaloids interaction with tubulin protein, interfere with the assembly of microtubules, leads to cell division arrest in metaphase. Similarly, vinflunine, a potent inhibitor of tubulin, causes hindrance in microtubule assembly and induces apoptosis. Moreover, vinflunine apoptosis mechanism involved activation of caspases 3 and 7 and c-Jun N-terminal kinase 1. 100 Other than vinca alkaloids; another alkaloid compound berberine induces cell cycle arrest and mitochondrial or intrinsic pathway in MCF-7 and MDA-MB-231 cells. 101,102 Similarly, noscapine induces apoptosis in breast cancer cells via intrinsic and extrinsic pathways by upregulation of Bax, downregulation of Bcl-2 and activation of caspases. [103][104][105] The hirsutine causes cell death in MDA-MB-231 cells by activating the intrinsic pathway of apoptosis and targeting NF-κB signaling pathway. 106,107 Moreover, the treatment of MCF-7 cells by procaine decreases DNA methylation and RARβ2 promoter methylation. 108

Terpenes
Terpenes or terpenoids classified based on the number of C5 units or cyclic structures present in the molecule. 109 The terpenoids can exert a broad spectrum of biological activities such as antioxidation, anti-inflammation, and anticancer activities. Numerous terpenoid compounds are known to possess anticancer potential in a verity of human cancers by causing inhibition of cancer cell proliferation and inducing apoptosis. Monoterpenoids such as D-limonene, have demonstrated antitumor and anticancer activities against breast cancer. 110 Several diterpenoids also possess anticancer activity against breast cancers and are involved in the induction of apoptosis. These include triptolide, oridonin, and ponicidin. 111 The triptolide also possesses antiproliferative activity and down-regulates the expression of ERα in different breast cancer cell lines. [112][113][114] The triterpenoids are close to steroids in structure and evoke apoptosis in a variety of cancer such as prostate and breast cancer. Different triterpenoids, like cucurbitacins, dammaranes, friedelanes, limonoids, lanostanes, lupanes, oleananes, tirucallanes, and ursanes, have been isolated from plants and studied for anticancer efficacy in breast cancer cells. 115 Ursolic acid, a triterpene acid causes DNA fragmentation induced apoptosis in MCF-7 cells by downregulation of Bcl-2 and activation of caspase -3. 116,117 Tetraterpenes also was known as carotenoids broadly categorized as acyclic tetraterpenoids and bicyclic tetraterpenoids. Carotenoids or tetraterpenoids such as lycopene and lutein are also known to possess anticancer activities in breast cancer cell lines. 111

Saponins
Saponins are natural glycosides widely distributed in plants classified into; triterpenoid, saponins, and steroid saponins. 118 Saponins possess potential biological activities includes; anti-inflammatory, antiproliferative, immunomodulatory, and anticancer activities. 119 Several saponins possess anticancer activities against various cancer cell lines. 120 For example, Avicin D, a triterpenoid glycoside compound, induces apoptosis in cutaneous T-cell lymphoma cells via downregulation of p-STAT-3 and bcl-2. 121 Similarly, tubeimoside-1 exhibits anticancer effects via mitochondrial dysfunction and endoplasmic reticulum stress pathways in HeLa cells. 122 The steroid saponins, degalactotigonin, and Polyphyllin D, possess cytotoxicity activity in ER+ human breast cancer cell line, MCF-7. 123,124 Moreover, the triterpene saponins such as gummiferaoside B and C possess antiproliferative activity in MDA-MB-435 cells. 125 Also, Avicins D, G induce apoptosis and cell cycle arrest in MDA-MB-435 cell line. 126 The phenolics, alkaloids, terpenoids and saponins derived from other sources also possess anticancer activity in different breast cancer cell lines. [127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142] Moreover, plant extracts, phytochemicals, and their potential mechanism of action against breast cancer are enlisted in Tables 1 and 2, respectively. Also, our various studies show the induction of cell death (apoptosis) in other cell lines. 143 The induction of cell death mainly due to presence of potential phytochemicals such as phenolics, saponins, terpenoids. The more screening and mechanistic studies need to be done to fully explore the potent phytochemicals in field of cancer therapeutics.

Conclusion
Cancer is a complex disease, leading cause of death worldwide. Despite the development of many synthetic anticancer drugs, toxicity remains the main problem, which reduces the survival rate. Therefore, there is an increase in demand for alternative treatments. Amongst the alternative approaches, the natural product derived anticancer agents are a practical choice. The secondary metabolites, as potential anticancer agents Table 1. Plant extracts and their potential mechanism of action against breast cancer