Abstract

Background

   Breast cancer is one of the most common female malignancies. This study
   explored the underlying mechanism through which the two plant compounds
   (Brucaine D and Narclasine) inhibited the proliferation of breast
   cancer cells.

Objective

   The purpose of this study was to explore the effect of Brucaine D and
   Narclasine on breast cancer development and their potential drug
   targets.

Methods

   [26]GSE85871 dataset containing 212 samples and the hallmark gene set
   “h.all.v2023.1.Hs.symbols.gmt” were downloaded from the Gene Expression
   Omnibus (GEO) database and the Molecular Signatures Database (MSigDB)
   database, respectively. Principal component analysis (PCA) was applied
   to classify clusters showing similar gene expression pattern. Single
   sample gene set enrichment analysis (ssGSEA) was used to calculate the
   hallmark score for different drug treatment groups. The expressions of
   genes related to angiogenesis, glycolysis and cell cycle were detected.
   Protein-protein interaction (PPI) network analysis was performed to
   study the interaction of the hub genes. Then, HERB database was
   employed to identify potential target genes for Narclasine and Bruceine
   D. Finally, in vitro experiments were conducted to validate partial
   drug-target pair.

Results

   PCA analysis showed that the significant changes in gene expression
   patterns took place in 6 drugs treatment groups (Narciclasine, Bruceine
   D, Japonicone A, 1beta-hydroxyalatolactone, Britanin, and four mixture
   drugs) in comparison to the remaining drug treatment groups. The ssGSEA
   pathway enrichment analysis demonstrated that Narciclasine and Bruceine
   treatments had similar enriched pathways, for instance, suppressed
   pathways related to angiogenesis, Glycolysis, and cell cycle, etc..
   Further gene expression analysis confirmed that Narciclasine and
   Bruceine had a strong ability to inhibit these cell cycle genes, and
   that MYC, CHEK2, MELK, CDK4 and EZH2 were closely interacted with each
   other in the PPI analysis. Drug target prediction revealed that
   Androgen Receptor (AR) and Estrogen Receptor 1 (ESR1) were the targets
   for Bruceine D, and Cytochrome P450 3A4 enzyme (CYP3A4) was the target
   for Narciclasine. Cell experiments also confirmed the connections
   between Narciclasine and CYP3A4.

Conclusion

   The present study uncovered that Narciclasine and Bruceine D could
   inhibit the growth of breast cancer and also predicted the potential
   targets for these two drugs, providing a new therapeutic direction for
   breast cancer patients.

Introduction

   Breast cancer as one of the most common female malignancies with a high
   heterogeneity [[27]1, [28]2] accounts for 25–30% of all malignancies
   among European and American women [[29]3], and its incidence has been
   increasing in recent years [[30]4]. Moreover, breast cancer is now the
   most frequent female tumor in developed regions of China [[31]5].
   Despite a variety of treatment options, the outcome and survival of
   breast cancer remains dismal due to its heterogeneous, invasive,
   recurrent and metastatic nature [[32]4]. A lack of biological targets
   and comprehensive understanding of the mechanism underlying the
   tumorigenesis restricts the development of therapeutic strategies for
   breast cancer [[33]6]. Conventional chemotherapies using taxanes,
   vinorelbine and anthracyclines are the primary treatments, particularly
   in preoperative setting [[34]7–[35]9]. In recent years, natural
   products have been increasingly used as potential anticancer drugs
   [[36]10]. Bruceine D and Narclasine are two natural products derived
   from plants that are believed to have anti-cancer ability [[37]11,
   [38]12]. CDK4 kinase is an important protein kinase in cell cycle
   regulation and is closely related to the tumorigenesis and progression
   of breast cancer. In this study, we explored the effects of Bruceine D
   and Narclasine on CDK4 and their role in regulating cell cycle in
   breast cancer.

   Cell cycle involves a series of complex events, through which a cell
   duplicates its contents and divides to produce two genetically
   identical daughter cells [[39]13]. Cell cycle and its regulation are
   crucial to cell growth and multiplication and involves regulatory
   proteins such as cyclin-dependent kinases (CDKs) and cyclin proteins,
   oncogenes and tumor-suppressor genes [[40]14]. Mitotic checkpoint
   protein allows cell cycle stages to proceed or to be inhibited
   [[41]14]. Cell cycle consists of interphases of G1 (beginning of S
   phase), S (DNA replication) and G2 (beginning of mitosis) phases, and
   mitotic (M) phase [[42]15]. During the whole cell cycle phase, cyclin
   proteins and CDKs form complexes and catalyze progression of cell cycle
   after activation and they are seen as the fundamental aspects of
   regulation [[43]16]. CDKs as key factors are only activated by cyclins
   at certain points of cell cycle [[44]17]. When any incomplete process
   or damage occurs in the cell cycle, cyclin-CDK regulatory activity will
   be blocked, resulting in arrested cell division cycle until these
   abnormalities are resolved [[45]18]. Some studies have found that three
   CDKs (CDK2, CDK4, and CDK6) are involved in interphase regulation,
   leading to exit from or entry into sub-phases [[46]16]. DNA damage
   signals inhibit these CDKs, inducing cell-cycle arrest [[47]19].
   Activation of CDK4 and CDK6 affects the progression of G1 early stage,
   then they bind with cyclin-D to phosphorylate the retinoblastoma tumor
   suppressor protein (RB) responsible for inactivating E2F transcription
   factors [[48]20]. E2F encoding proteins are necessary for the G1/S
   transition and facilitates cells to the next cycle phases [[49]21].
   CDK4 and CDK6 inhibited by DNA damage detection cannot inactivate RB,
   which allows RB to bind to E2F and further suppress the activity of
   E2F, thereby leading to cell cycle arrest [[50]16]. In breast cancer,
   overactivation of CDK4 causes abnormal cell cycles that promote the
   proliferation and metastasis of tumor cells [[51]22]. Therefore,
   inhibition of CDK4 activity has become an important strategy for
   treating breast cancer.

   Bruceine D and Narclasine are two natural products isolated from the
   shrub Brucea javanica (L.) Merr. (Simaroubaceae) [[52]23] and the
   Narcissus species (Amaryllidaceae) [[53]12], respectively. Recent
   studies have shown that these two compounds have antitumor activity.
   Bruceine D could inhibit the proliferation and invasion of
   hepatocellular carcinoma [[54]24] and induce apoptosis and autophagy of
   lung cancer [[55]25]. Narclasine has anti-proliferative and
   anti-invasive effects on a variety of cancer cells [[56]12], for
   example, it inhibits esophageal cancer cell proliferation and migration
   via suppressing FAK signaling pathway [[57]26]. However, the specific
   mechanisms through which these two compounds regulate CDK4 signaling
   and their role in regulating breast cancer cell cycle remained unclear.
   To bridge such a gap, the present study investigated the effects of the
   two compounds on CDK4 kinase activity and their regulatory mechanisms
   in breast cancer cell cycle applying bioinformatics analysis, hoping to
   contribute to the treatment of breast cancer.

Material and methods

Data acquisition

   The RNA-sequencing data from 212 human breast cancer cell line (MCF7)
   samples treated by 102 drugs were downloaded from GEO (GEO:
   [58]GSE85871, [59]https://www.ncbi.nlm.nih.gov/geo/) database [[60]27].

Data preprocessing

   The expression matrix was read by the GEOquerry R package and the box
   plot was used to assess differences or abnormal value in data [[61]28].
   Then, the mean value in the same group was taken as the gene expression
   level to perform the Principal Component Analysis (PCA) for
   dimensionality reduction. Drug-treated groups showing significant
   changes in expression patterns were extracted from the gene expression
   data.

Hallmark enrichment score analysis

   The hallmark gene set “h.all.v2023.1.Hs.symbols.gmt” was downloaded
   from MSigDB
   ([62]https://www.gsea-msigdb.org/gsea/msigdb/collections.jsp) database
   [[63]29] and used to compute the hallmark enrichment score of each
   sample by the single sample gene set enrichment analysis (ssGSEA)
   algorithm using GSVA R package [[64]30].

Screening differentially expressed genes (DEGs)

   The mean values of gene expression calculated by the t-test were used
   to indicate the up- or down-regulation in genes from different groups.
   DEGs were screened under p value < 0.05.

Protein-protein interaction (PPI) network analysis

   The screened hub genes were subjected to PPI networks analysis, and the
   degree of each gene was calculated by the CytoNCA package in Cytscape
   software (version 3.8.0), which could be applied to evaluate the
   connectivity degree of nodes and identify the most important clusters
   of nodes in a network [[65]31].

Drug target prediction

   HERB database [[66]32] ([67]http://herb.ac.cn/Search/) was used to
   predict the target for the drugs of Bruceine D and Narclasine. Briefly,
   we entered the name of Bruceine D and Narclasine in the column of
   “Ingredient” and clicked the “Search” button for analysis.

Cell culture and drug treatment

   Neoplastic MCF7 and non-neoplastic MCF10A cell lines were purchased
   from American Type Culture Collection (ATCC, Manassas, VA, USA) and
   maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich,
   USA) supplemented with 10% Fetal Bovine Serum (FBS, Sigma-Aldrich,
   USA). Narciclasine of ≥ 98% purity was purchased from Biopurify
   Phytochemical (Chengdu, China). Narciclasine stock solution was
   prepared applying dimethyl sulphoxide (DMSO) and diluted to final the
   concentration in culture medium. Accordance to a recently published
   paper [[68]33], MCF-7 cells were treated with Nar (100 nM) for 24 h.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

   Total mRNA was extracted using the TRIzol reagent (Invitrogen)
   following the manufacturer’s instruction. QRT-PCR amplifications were
   conducted adopting the FastStart Essential DNA Green Master and
   LightCycler 96 Instrument (Roche, Basel, Switzerland). Total mRNA was
   reverse-transcribed into cDNA using a PrimeScript RT reagent kit
   (Takara, Shiga, Japan). The PCR was amplified under the following
   conditions: 50 cycles of 94°C for 10 minutes, 94°C for 10 seconds, and
   55°C for 45 seconds. The primer sequences were designed as follows:
   CYP3A4: forward 5′-GGTGGTGGTGATGATTCC-3′ and reverse
   5′-TTGAAGAAGTCCTCCTAAGC-3′; GAPDH: forward 5′- AATGGGCAGCCGTTAGGAAA-3′
   and reverse 5′- GCCCAATACGACCAAATCAGAG-3′.

Statistical analysis

   All statistical analysis and visualization were performed using the R
   software (version 4.3.1). The students test was used to compare the
   differences between two sets of continuous variables. P value < 0.05
   was regarded as statistical difference. Some supporting analyses were
   provided by Sangerbox ([69]http://sangerbox.com/home.html).

Results

Gene expression pattern of MCF7 cell lines after drug treatment

   Visualization of the expression levels of all genes in each sample
   ([70]Fig 1A) showed that the gene expressions were evenly distributed,
   therefore no normalization was required. PCA analysis revealed that the
   gene expression pattern had significant changes in the Narciclasine,
   Bruceine D, Japonicone A, 1beta-hydroxyalatolactone, Britanin and
   Four-mixture (tanshinone IIA, salvianic acid A sodium, protocatechuic
   aldehyde, salvianolic acid B) groups compared with other drug treatment
   groups ([71]Fig 1B). The 2D structure of the 5 pure chemical compound
   drugs were shown in [72]Fig 1C. These groups with similar gene
   expression patterns were named as common groups, and we further
   explored the differences in pharmacological molecular mechanisms
   between the significantly changed groups (including six drugs) and
   common groups.

Fig 1. The expression pattern of MCF7 cell line after drugs treatment.

   [73]Fig 1
   [74]Open in a new tab

   (A) The box plot of expression matrix in MCF7 cell lines treated by
   drugs. (B) PCA analysis of MCF7 cell lines treated by drugs. (C) The 2D
   structure of the 5 pure chemical compounds.

Expression profile difference analysis on MCF7 cell lines after drug
treatment

   To explore the effect of the 6 drugs on the expression profile of MCF7
   cell lines, we calculated the ssGSEA score in the corresponding groups
   in the Hallmark gene set. Specifically, the enrichment pathway in the
   Narciclasine and Bruceine D treatment groups was similar ([75]Fig 2A
   and 2B), the pathways of Angiogenesis, Glycolysis, G2M checkpoint, Myc
   targets v1 and E2F targets were suppressed, and the p53, TNFA signaling
   via NFKB, apoptosis and inflammatory response pathway were activated.
   The pathways of Glycolysis, Interferon gamma/alpha response in
   1beta-hydroxyalatolactone treatment groups were inhibited, while p53
   and heme metabolism pathways were activated ([76]Fig 2C). Japonicone A
   suppressed the expression of G2M checkpoint, Myc targets v1, and E2F
   targets genes ([77]Fig 2D). Britanin suppressed the expression of E2F
   targets and G2M checkpoint genes ([78]Fig 2E). Four mixture drugs
   inhibited the expressions of the target gene for E2F ([79]Fig 2F).
   Combined with the results from PCA clustering and pathway enrichment
   analysis, we found that tNarciclasine and Bruceine D had similar
   anti-cancer mechanisms and were distinct from other drug treatment
   groups. Thus, the pharmacological mechanisms of these two drugs were
   further studied.

Fig 2. Expression profile feature of MCF7 cell line after drugs treatment.

   [80]Fig 2
   [81]Open in a new tab

   (A) Enrichment scores of MCF7 cells treated by Narciclasine. (B)
   Enrichment scores of MCF7 cells treated by Bruceine D. (C) Enrichment
   scores of MCF7 cells treated by Japonicone A. (D) Enrichment scores of
   MCF7 cells treated by 1beta-hydroxyalatolactone. (E) Enrichment scores
   of MCF7 cells treated by Britanine. (F) Enrichment scores of MCF7 cells
   treated by four mixture (tanshinone IIA, salvianic acid A sodium,
   protocatechuic aldehyde, salvianolic acid B).

The angiogenesis and glycolysis were suppressed by Narciclasine and Bruceine
D

   Significantly down-regulated DEGs between Narciclasine and Bruceine D
   treatment groups were screened. As angiogenesis and glycolysis play an
   important role in tumor development, increase in glycolysis is a major
   hallmark of tumor progression and can help tumor cells obtain more
   energy in an oxygen-free environment [[82]34]. Abnormally increased
   angiogenesis could provide nutrients and oxygen to support cell growth
   and proliferation of tumors [[83]35] and, in turn, lactic acid produced
   by glycolysis can promote angiogenesis [[84]36]. Thus, down-regulated
   DEGs related to angiogenesis and glycolysis were analyzed.
   Angiogenesis-related DEGs including the APP, KCNJ8, SERNIPA5, STC1 and
   TNFRSF21 were significantly down-regulated ([85]Fig 3A and 3B), and
   glycolysis-related DEGs including ANKZF1, HSPA5, IDH1, LDHA, MXI1,
   NT5E, PAM, PDK3, PGAM1, PGK1, STC1, STMN1 and TGFBI were significantly
   down-regulated ([86]Fig 3C). Interestingly, Venn plots of angiogenesis-
   ([87]Fig 3D) and Glycolysis-related genes ([88]Fig 3E) showed that the
   STC1 co-participated in the inhibition of angiogenesis and glycolysis
   of the two drugs. Previous studies reported that stanniocalcin-1 (SCT1)
   acted as a tumor growth factor to promote tumor proliferation, and that
   overexpression of SCT1 can promote tumor proliferation and subcutaneous
   tumor formation in mice [[89]37], whereas inhibition of SCT1 reduced
   the tumor cell proliferation [[90]38]. Increasing studies indicated
   that the effect of STC1 on promoting cell proliferation was closely
   associated with cell cycle changes. In ovarian cancer, STC1 stimulated
   high expression of cell cycle-related proteins (cyclin A/B1/D1 and
   CDK2/4) for rapid proliferation of tumor cells [[91]39] and mitotic
   cycle of G1 to S was dramatically shortened [[92]40]. In addition, STC1
   also activates ERK and JNK pathway promoting inhibitor of apoptosis
   proteins (BcL-2 and BcL-xl) and inhibits the expression of
   pro-apoptotic proteins (Bax, Bak and Bid) for tumor survival [[93]41].
   These findings suggested that Narciclasine and Bruceine D could inhibit
   the proliferation of MCF7 cells through regulating cell cycle.

Fig 3. The expression of angiogenesis and glycolysis related genes in
comparing with common groups.

   [94]Fig 3
   [95]Open in a new tab

   (A) The expression levels of angiogenesis-related genes in MCF7 cells
   that treated by Narciclasine. (B) The expression levels of
   angiogenesis-related genes in MCF7 cells that treated by Bruceine D.
   (C) The expression levels of glycolysis-related genes in MCF7 cells
   that treated by Narciclasine and Bruceine D. (D) Venn plot of
   angiogenesis-related genes in Narciclasine and Bruceine D treatment
   groups. (E) Venn plot of glycolysis-related genes in Narciclasine and
   Bruceine D treatment groups. *p < 0.05, ***p < 0.001, ****p < 0.0001.
   The vertical axis in Fig C and D means the expression level of genes,
   where the red square represents high expression, and the blue square
   represents low expression.

Narciclasine and Bruceine D inhibited tumor proliferation through regulating
cell cycle

   Next, we investigated pathway related to cell cycle to further explore
   the mechanism through which Narciclasine and Bruceine D inhibited the
   proliferation of breast cancer cells. The expression profile in the G2M
   checkpoint, E2F target, MYC target v1 pathway were analyzed. Tumor
   cells are cells with DNA defects. Continuous activation of G2M
   checkpoint arrests tumor cells in G2 phase [[96]42]. Anti-cancer
   strategies based on G2M checkpoints focus on targeting G2M checkpoint
   inactivation, forcing cancer cells to enter mitotic M phase, which
   ultimately leads to mitotic catastrophe and cell death [[97]43]. The
   expression of E2F target genes is gradually up-regulated during G1,
   promoting DNA reproduction and cell division from the G1 to S stage
   [[98]44]. MYC targets are a set of nucleoprotein-like oncogenes that
   regulate the expression of multiple genes, promote cell proliferation
   and metastasis, and inhibit cell apoptosis [[99]45]. The expression of
   genes related to G2M checkpoint was down-regulated ([100]Fig 4A and
   4B), including CHEK2, MELK, CDK4. The transcription factors MYC and
   methyltransferase EZH2 with cell cycle regulation role in E2F targets
   pathway were down-regulated in the Narciclasine and Bruceine D
   treatment groups ([101]Fig 4C). However, the genes associated with MYC
   target v1 pathway were not obviously involved in cell cycle regulation
   ([102]Fig 4D). In the PPI network analysis, PBK and GINS2 were two
   shared genes in G2M checkpoint pathway ([103]Fig 5A) between
   Narciclasine and Bruceine D, and the PBK had the highest connectivity
   degree with other genes through PPI analysis ([104]Fig 5B and 5C).
   Narciclasine and Bruceine D in E2F target pathway had 25 shared genes
   including CHEK2, MELK, CDK4 ([105]Fig 5D), which also possessed higher
   connectivity degree with other genes ([106]Fig 5E and 5F). The MYC
   target v1 pathway had 9 shared genes ([107]Fig 5G), with TYMS having
   the highest connectivity degree with other genes ([108]Fig 5H and 5I).
   The high connectivity degree, the more importance of the genes and
   related pathways. Thus, we speculated that Narciclasine and Bruceine D
   could inhibit MCF7 cell proliferation through targeting MELK, CDK4, and
   MYC to regulate cell cycle in breast cancer. PPI analysis also
   disclosed a close connection between these 5 genes ([109]Fig 6A), and
   some of these genes were in a hub position of cell cycle process from
   KEGG database ([110]Fig 6B).

Fig 4. The expression of cell cycle related genes in comparing with common
groups.

   [111]Fig 4
   [112]Open in a new tab

   (A) The expression of genes related to G2M checkpoints pathway in MCF7
   cells treated by Bruceine D. (B) The expression of genes related to G2M
   checkpoints pathway in MCF7 cells treated by Narciclasine. (C) The
   expression of genes related to E2F targets pathway in MCF7 cells
   treated by Narciclasine and Bruceine D. (D) The expression of genes
   related to MYC targets pathway in MCF7 cells treated by Narciclasine
   and Bruceine D. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
   The vertical axis in Fig C and D means the expression level of genes,
   where the red square represents high expression, and the blue square
   represents low expression.

Fig 5. The shared genes between Narciclasine and Bruceine D treatment groups.

   [113]Fig 5
   [114]Open in a new tab

   (A) G2M checkpoints genes shared by Bruceine D and Narciclasine in MCF7
   cells. (B) The connectivity degree of G2M checkpoints gene in
   Narciclasine treatment group. (C) The connectivity degree of G2M
   checkpoints gene in Bruceine D treatment group. (D) E2F target genes
   shared by Bruceine D and Narciclasine in MCF7 cells. (E) The
   connectivity degree of E2F target genes in Narciclasine treatment
   group. (F) The connectivity degree of E2F target genes in Bruceine D
   treatment group. (G) MYC target genes shared by Bruceine D and
   Narciclasine in MCF7 cells. (H) The connectivity degree of MYC target
   genes in Narciclasine treatment group. (I) The connectivity degree of
   MYC target genes in Narciclasine treatment group.

Fig 6. PPI analysis of 5 cell cycle related genes.

   [115]Fig 6
   [116]Open in a new tab

   (A) PPI network analysis of key cell cycle genes. (B) The position of
   some hub genes in cell cycle process.

The potential drug targets for Narciclasine and Bruceine D and partial
validation

   Drug prediction analysis uncovered that Androgen Receptor (AR) and
   Estrogen Receptor 1 (ESR1) were the targets for Bruceine D, and that
   Cytochrome P450 3A4 enzyme (CYP3A4) was the target for Narciclasine
   ([117]Fig 7A). Next, we carried out in vitro experiments to confirm the
   connections of Narciclasine and CYP3A4. We first detected the
   expressions of CYP3A4 in normal and tumor breast cell lines. As shown
   in [118]Fig 7B, the expression of CYP3A4 was up-regulated in MCF 7
   cells in comparison to normal MCF 10A cells but it was significantly
   down-regulated when MCF 7 cells were treated with 50nM Narciclasine
   ([119]Fig 7C). These results validated that CYP3A4 was the target for
   Narciclasine.

Fig 7. Drug target prediction and validation.

   [120]Fig 7
   [121]Open in a new tab

   (A) Predicted drug target for Narciclasine and Bruceine D. (B) The mRNA
   levels of CYP3A4 in normal (MCF 10A) and tumor (MCF 7) breast cells.
   (C) The mRNA levels of CYP3A4 in tumor (MCF 7) breast cells after
   Narciclasine treatment. N = 3, ** P < 0.01. The results are presented
   as mean ± SEM.

Discussion

   Increasing findings support the use of Narciclasine and Bruceine D as
   antitumor agents [[122]33, [123]46]. Several studies have reported the
   antitumor mechanisms of the two drugs [[124]47–[125]49], but natural
   products often have multiple molecular targets. Therefore, based on the
   transcriptome data of 212 breast cell lines treated by different drugs,
   this study comprehensively explored the underlying antitumor targets
   for Narciclasine and Bruceine D.

   Breast cancer is one of the most common malignant tumors to women,
   especially, hormone receptor-positive breast cancer [[126]50] accounts
   for 60%-70% of all breast cancer cases [[127]51] and endocrine therapy
   is currently the main treatment for this type of breast cancer
   [[128]52]. Though treatments for the cancer have been greatly improved,
   a considerable number of patients will eventually develop resistance to
   endocrine therapy [[129]53], leading to a poor prognosis. Previous
   studies indicated that endocrine resistance is resulted from multiple
   mechanisms, for instance, loss or mutation in estrogen receptor (ER)
   and deregulation of cell cycle signaling molecules [[130]54, [131]55].
   Thus, researchers identified novel therapeutic targets based on these
   mechanisms, including cyclin-dependent kinase (CDK) 4/6 inhibitors.
   CDK4/6 is involved in the transition from G1 to S phase, the complex
   cyclin D- CDK4/6 phosphorylates RB and dissociates them from the E2F to
   promote cell cycle progression [[132]56]. There are various factors
   that can cause hyperphosphorylation of RB, for example, cyclin D is
   overexpressed in about half of breast cancers and this may ultimately
   lead to uncontrolled cell proliferation [[133]57]. The pathway
   CDK-RB1-E2F targeted by CDK4/6 inhibitors is vital for inhibiting the
   proliferation of tumor cells and it is disrupted in a majority of
   cancers [[134]58, [135]59]. Currently, palbociclib, ribociclib and
   abemaciclib as three CDK 4/6 inhibitors [[136]60] that have remarkable
   effect when used in combination with endocrine therapies and they have
   all been approved by Food and Drug Administration (FDA) [[137]61].

   In addition, PBK is a shared down-regulated genes with the highest
   connectivity degrees in the Narciclasine and Bruceine D treatment
   groups, indicating that it played important role in breast cancer cell
   proliferation. PBK is a serine/threonine kinase that is considered as
   oncogenic protein involved in cell growth, proliferation, apoptosis and
   inflammation [[138]62, [139]63]. PBK is aberrantly overexpressed in
   many cancers, such as adrenocortical carcinoma [[140]64],
   hepatocellular carcinoma [[141]65], and pancreatic cancer [[142]66].
   PBK has also been reported as a mitosis kinase activated by the complex
   of cyclin B1-CDK1 to promote cytokinesis through the phosphorylation of
   polycomb repressive complex 1(PRC1) [[143]67]. PBK binding to PRC1
   increases the phosphorylation of PRC1 at T481 site, which further
   elevates the phosphorylation level of cyclin B1-CDK1 to PRC1 and
   ultimately promotes cell cycle division [[144]67]. Previous studies
   indicated that overexpression of PBK increases the cell proliferation
   and tumorigenesis in JB6 epidermal cells in vivo and in vitro [[145]68,
   [146]69], while knockdown of PBK suppresses tumor growth of lung cancer
   cell lines [[147]70]. Some studies also revealed that the expression
   levels of PBK and kinase activity are positively correlated with the
   G2/M phase cell numbers in prostate cancer and gastric carcinoma.
   Silencing or inhibiting the enzymatic activity of PBK can arrest tumor
   cells in G0 phase [[148]71] because the PBK-CDK1-cyclin B complex could
   prolong the degradation of cyclin B [[149]72]. Together, these findings
   indicates the inhibitory effect of Narciclasine and Bruceine D on cell
   cycle to block the tumor proliferation of breast cancer.

   In this study, Narciclasine and Bruceine D showing similar pathway
   enrichment were screened by target prediction analysis. AR and ESR1
   were the targets for Bruceine D, and CYP3A4 was the target for
   Narciclasine. AR is a type I nuclear receptor that regulates gene
   transcription for cell differentiation, proliferation, apoptosis, or
   angiogenesis of breast cancer [[150]73]. The fact that AR levels exceed
   70% in both primary and metastatic breast cancer suggests that AR could
   be a novel therapeutic target for AR+ breast cancer patients [[151]74].
   ESR1 is responsible for encoding estrogen receptor α (ER). Although
   targeting of ER+ breast cancer patients with endocrine therapy (ET) is
   currently a standard treatment [[152]75], mutations of ESR1 is the
   essential driver of ET resistance [[153]74]. Therefore, in-depth
   studies should be performed to probe into the effect of Bruceine D on
   mutated ESR1 in breast cancer. Carbon Monoxide can suppress the levels
   of CYP3A4 to enhance the sensitivity of human breast cancer cells to
   Paclitaxel [[154]76]. In this research, we found that a high level of
   CYP3A4 in MCF 7 cells could be reduced by Narciclasine treatment,
   suggesting a new target drug for breast cancer treatment. In short,
   drug prediction results showed a potential therapeutic direction for
   breast cancer.

Conclusion

   We performed a comprehensive analysis of breast cancer cells treated by
   different drugs and identified several drug targets for both
   Narciclasine and Bruceine D. These two drugs can inhibit the growth and
   proliferation of breast cancer cells. The current findings are expected
   to contribute to the clinical development of potential therapies for
   breast cancer patients.

Abbreviations

   AR
          Androgen Receptor

   CDK4
          cyclin-dependent kinase 4

   CYP3A4
          Cytochrome P450 3A4 enzyme

   ER
          Estrogen receptor α

   ESR1
          Estrogen Receptor 1

   ET
          endocrine therapyGEO, Gene Expression Omnibus

   ssGSEA
          single sample gene set enrichment analysis

   PPI
          Protein-Protein Interaction

   PCA
          Principal Component Analysis

   MCF7
          human breast cancer cell lines

   MSigDB
          Molecular Signatures Database

   DEGs
          Differentially expressed genes

   SCT1
          stanniocalcin-1

Data Availability

   The datasets generated and/or analyzed during the current study are
   available in the [GSE85871] repository,
   [[155]https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE85871].

Funding Statement

   The author(s) received no specific funding for this work.

References