Research Progress of Plant-Derived Natural Products against Drug-Resistant Cancer
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript presents an interesting approach regarding the ability of some plant principles to be used in antitumor therapy as an alternative to drug resistant cancer. However, there is still a long way to go until their introduction into the current therapy for the treatment of cancer, they are still at the stage of alternative therapy due to the many impediments that make it difficult to isolate and properly test these active principles.
In my opinion, the manuscript should be a major revision.
As comments/suggestions:
1. The introduction is too brief and does not present clear objectives with outstanding scientific evidence for the use of natural products in the fight against resistance to conventional antitumor therapies.
2. The presentation of the mechanisms by which resistance is established to the current antitumor therapies must be accompanied by examples of existing products on the market for which this conclusion was reached.
3.The studies of the potential mechanisms of action of natural products were done using what antitumor as a reference?
4. Is there any compound of the type of natural plant extracts that has reached clinical trials for the treatment of a certain type of cancer?
Author Response
Dear Editors and Reviewers,
Thanks for your letter concerning our manuscript entitled “Research Progress of Plant-Derived Natural Products against Drug Resistant Cancer”. We tried our best to improve the manuscript and made some changes to the manuscript. These changes will not influence the content and framework of the paper. Once again, thank you very much for your comments and suggestions.
Reviewer(s)' Comments to Author:
Reviewer: 1
Comments to the Author
1 The introduction is too brief and does not present clear objectives with outstanding scientific evidence for the use of natural products in the fight against resistance to conventional antitumor therapies.
R: Thank you for your positive suggestion. We have supplemented the introduction with additional evidence that natural products can be used to fight against resistance to conventional antitumor therapies. The modifications are as follows(see online 31 to 69 in paper）:
Cancer is one of the most dominant cause of death in the world. It is estimated that more than 19.3 million new cases have been diagnosed with 10 million deaths annually worldwide [1]. The gender gap for the incidence rate of all cancers is twice, with 19% higher in men than in women in 2020, although differences in the distribution of widely across regions [2]. Moreover, the burden of cancer incidence and mortality is rapidly growing around the world. Currently, the main cancer treatment strategies include surgical resection [3], radiotherapy, chemotherapy [4], immunotherapy [5] and targeted therapy [6]. Although the conventional therapies are effective at early stage of cancer, they have limited efficacy for locally occurred and metastatic cancer due to severe side effect, drug resistance, multiple recurrences and metastases [7, 8, 9].
A major reason for treatment failure in cancer patients is the resistance to chemo-therapeutic agents [10]. Drug resistance to therapies in cancer can be classified as primary or acquired. Primary resistance refers to the ability of cancers to escape initial therapy. And acquired drug resistance develops after continuous exposure to chemotherapeutic drug, even though the drug is initially active [11]. Body gains a cross-resistance to variety of chemotherapeutic drugs with different cellular targets and functions, which is called multiple drug resistance (MDR) [12]. It is ineffective and toxic that using high doses of drugs to overcome multidrug resistance [13]. Therefore, there is an urgent need to de-velop therapeutic agents with a better safety profile and higher efficacy for drug resistant cancer treatment.
For the past few years, plant-derived natural products have been evaluated as the most potential candidates for drug resistant oncology therapies. They can overcome drug resistance with low side effect. A variety of plant-derived natural products with an-ti-drug-resistant-tumor activities have been identified, such as alkaloids, terpenoids, phenols, flavonoids, which can inhibit expression of resistant protein [14], suppress tu-mor-cell invasion and migration [15], induce apoptosis [16], restrain angiogenesis and proliferation [17]. Studies have demonstrated that flavonoids in combination with suita-ble anticancer drugs have improve their therapeutic index by increasing their bioavaila-bility, thereby reducing lethal side effects by lowering the dose of chemotherapeutic agents [18]. It was proved that MDR in gastric cancer reversed by curcumin is closely related to NF-κB-mediated apoptosis [19，20], and the chinese herb Glaucine, an Iso-quinoline alkaloid isolated from the stem of Corydalis yanhusuo, caninhibit P-gp and MRP1-mediated efflux and increase ATPase activity of the transporter protein pumps in drug resistant breast cancer cells MCF-7/ADR [21]. β-Elemene can attenuate SGC7901 resistance to VCR and ADM by decreasing p-gp and MRP, and attenuates exo-some-mediated resistance and metastasis in the multidrug-resistant gastric cancer cell line SGC7901/ADR[23]. In this review, we summarize the mechanisms of cancer drug re-sistance, as well as the anti-drug-resistant-cancer effects of plant-derived natural prod-ucts and underlying mechanisms, in order to provide a potent therapeutic strategy for drug-resistant tumors.
2 The presentation of the mechanisms by which resistance is established to the current antitumor therapies must be accompanied by examples of existing products on the market for which this conclusion was reached.
R: Thank you for your positive suggestion. We have added the mechanisms by which the current products available on the market for anti-tumor therapies generate resistance. The modifications are as follows(see online 81 to 114 in paper）:
Tumor heterogeneity is one of the reasons accounting for the different clinical out-comes of chemotherapy and targeted therapy in cancer patients. Genetic heterogeneity of tumor cells lead to different sensitivities to the various therapeutic agents. Tumor cells which exhibit low sensitivity to chemotherapy drugs bring about drug dissociation and form primary drug resistance [24]. On the other hand, acquired drug resistance de-velops after long-term exposure of chemotherapy drug to tumor cells that initially re-sponse to therapy [25]. Many factors that modulate the binding of chemotherapy drugs to tumor cells, DNA damage or apoptosis cause emergence of drug resistance [26]. Mechanisms of multiple drug resistance to cancer therapies are complex. Camptothecin (CPT) is extracted mainly from the dove tree, family Hippophae, which is endemic to China. The CPTs including topotecan (TPT), irinotecan (CPT-11), and 10-hydroxycamptothecin (HCPT), etc, have been marketed and widely used in clinical anticancer therapy. Among them, CPT-11 ,a pre-drug, is converted into the highly active metabolite SN-38 in vivo by the action of carboxylesterases. The current mechanisms of resistance to CPTs include (1) the activation of drug-transporting proteins such as the p-gp protein which excrete intracellular drugs out of the body; (2) Alteration of the target of drug action or enhancement of the target's restorative effect. (3) inhibition of apoptosis and cell cycle blockade[27]. Chemotherapeutic agents are currently categorized into two groups based on their origins. One is of compoundsextracted from plants [28,29] and another is synthetic compounds [30,31]. According to the mechanism of action, the synthetic ones can be categorized as alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic spindle inhibitors, etc, For example, pyrimidine antagonists (5-fluorouracil (5-FU), gemcitabine, and capecitabine) as the antimetabolites; methotrexate, pemetrexed, and pramlintide as antifolates, and hydroxyurea as ribonucleotide reductase inhibitors . These anticancer drugs interfere with important biosynthetic pathways, inhinder DNA/RNA synthesis or cause DNA strand breaks by inhibiting specific enzymes (dihydrofolate reductase, ribonucleotide reductase, and DNA polymerase) or by adulterating DNA with faulty structural analogs of pyrimidines/purines [30,32,33]. Cisplatin is a DNA intercalating agent that crosslinks DNA, thereby inhibit RNA transcription and DNA replication activities. If DNA damage is not repaired promptly, cell cycle arrest and apoptosis are triggered [34,35]. Cells can develop resistance to cisplatin through a variety of mecha-nisms, including alteration of intracellular drug accumulation by inhibition of uptake or enhanced efflux, detoxification of the drug by redox mechanisms, enhanced DNA exci-sion repair or negative regulation of apoptotic signaling [36-39].
3 The studies of the potential mechanisms of action of natural products were done using what antitumor as a reference?
R: Thank you for your positive suggestion. At present, there are about 80 common clinical antitumor drugs in the international arena, which can be roughly divided into the following six categories: cytotoxic drugs, hormonal drugs, biological response modifiers, monoclonal antibody drugs, other drugs, and adjuvants. For example, the nitrogen mustard alkylating agent-cyclophosphamide alkylates S-phase DNA in tumor cells and inhibits the growth of tumor cells; the dihydrofolate reductase inhibitor-methotrexate prevents the reduction of dihydrofolate to tetrahydrofolate by competitively inhibiting the enzyme dihydrofolate reductase and irreversibly combining with it, which inhibits the synthesis of purine nucleotides and pyrimidine nucleotides and makes the synthesis of deoxythymidine blocked, thus interrupting the synthesis of DNA and RNA, and blocking the reproduction of cancer cells. Natural products of plant origin have many advantages such as abundant resources, low toxicity, and diverse targets and molecular mechanisms. Recent studies have been based on the molecular mechanisms of chemotherapeutic drugs to find more effective low-toxicity natural products.
[1]. Sun Yan. Consensus recommendations on the classification of antitumor drugs[J]. Evidence-Based Medicine, 2004, 4(3):190-191.
4 Is there any compound of the type of natural plant extracts that has reached clinical trials for the treatment of a certain type of cancer?
R: Thank you for your positive suggestion. We have added some evidence of the compound of the type of natural plant extracts that has reached clinical trials for the treatment of a certain type of cancer. The modifications are as follows (see online 1095 to 1117 and references 151-153,174-177 in paper）
PTX, vincristine, vinorelbine, colchicine, epigallocatechin (etoposide and teniposide), Camptothecin (topotecan and irinotecan), curcumin, serpentine (dihydromyricetin), leucocephalos, capsaicin, and bittersweet have been reported to have efficacy in certain types of cancer. Paclitaxel is an anti-mitotic tumor drug, which has the effect of promoting the cohesion of microtubule protein and fixing microtubules. Elemene is an anti-cancer traditional Chinese medicine with independent intellectual property rights in China, which can inhibit tumor growth and induce differentiation and apoptosis, and it not only has direct anti-cancer effect, but also has immunomodulatory effect. It was found that Camptothecin, Irinotecan, and Topotecan were able to prevent single-strand breaks by stabilizing the topoisomerase I-DNA complex, which leads to DNA double-strand breaks; paclitaxel was able to inhibit microtubule function, which leads to cell-cycle arrest; and vincristine was able to inhibit microtubule polymerization and assembly, which leads to cell-cycle arrest and death. There are currently 13 natural antitumor drugs on the market, including paclitaxel, and more natural products and their derivatives need to be developed to continue to make significant contributions to human health and longevity.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
1. The paper does not provide a comprehensive discussion on the specific limitations of the research conducted.
2. The paper does not mention any potential limitations in terms of the methodology used or the reliability of the findings.
3. The paper does not address any potential biases or limitations in the selection of plant-derived natural products for evaluation.
4. The paper does not discuss any limitations in terms of the generalizability of the findings to different types of drug-resistant cancers.
5. The paper does not mention any limitations in terms of the translation of the research findings into clinical applications or the development of anti-resistant-cancer drugs.
6. The discussion should be rather organized around arguments avoiding simply describing details without providing much meaning.
7. Alkaloids and Terpenoids in Overcoming Drug Resistance: Need to add this section with mechanistic figure.
8. Polyphenols and Flavonoids as Reversal Agents: Need to add this section with mechanistic figure.
9. Synergistic Effects and Combination Therapies: Need to add this section.
10. Preclinical and Clinical Studies: Need to add this section.
11. Identification of challenges and limitations associated with the development and clinical translation of plant-derived natural products for overcoming drug resistance in cancer: Need to add this section.
12. Future Directions and Therapeutic Potential: Need to add this section.
Comments on the Quality of English Language
Major revisions
Author Response
Dear Editors and Reviewers,
Thanks for your letter concerning our manuscript entitled “Research Progress of Plant-Derived Natural Products against Drug Resistant Cancer”. We tried our best to improve the manuscript and made some changes to the manuscript. These changes will not influence the content and framework of the paper. Once again, thank you very much for your comments and suggestions.
Reviewer(s)' Comments to Author:
Reviewer: 2
Comments to the Author
1 The paper does not provide a comprehensive discussion on the specific limitations of the research conducted.
R: Thank you for your positive suggestion. We added the limitations of the study with additional changes to the discussion section. The modifications are as follows (see online 1079 to 1115 in paper）:
Natural products with plant origin have many advantages such as abundant resources, low toxicity, and diverse targets with various molecular mechanisms. The diversity of molecular structure and unique action mode of biological activity make them play an important role in the development of candidates for drug resistant cancer treatment . It is necessary to find some delivery strategies, such as exosomes or nanotechnology, in an attempt to overcome come these limitations and rediscover the new benefits associated with these natural products. In the study of drug resistance mechanisms, most of the current studies have focused on the classical mechanisms of the MDR gene and the P-gp encoded by the MDR gene, while few studies have focused on non-classical mechanisms such as GST-π, Topo II, DNA damage repair and tumor stem cells. In contrast, these mechanisms are rarely explored and relatively understudied in depth. In addition, most studies on cancer drug resistance mechanisms have focused on the cellular level and experimental animals.
2 The paper does not mention any potential limitations in terms of the methodology used or the reliability of the findings.
R: Thank you for your positive suggestion. Possible limitations in the reliability of the methods or findings used in the paper have been added., The modifications are as follows (see online 70 to 78 in paper）:
The review process is divided into three main steps: title, abstract, and content screening. This paper reviews previous studies of natural products on tumor drug resistance. Articles were searched in PubMed, CNKI, Web of science, X-mol and Springer databases. Search terms: plant-derived natural products; cancer; drug resistant; molecular mechanisms All titles were screened and 1354 documents were downloaded for abstract screening. After completing the initial screening, out of the 578 articles that met the inclusion criteria, finally, the full text of all 578 retained literature was critically evaluated to avoid duplication, leaving 296 papers to be included in this review.
3 The paper does not address any potential biases or limitations in the selection of plant-derived natural products for evaluation.
R: Thank you for your positive suggestion. This article has added additions to the biases or limitations that may exist when selecting plant-derived natural products for evaluation. The modifications are as follows(see online 1079 to 1092 in paper）:
Natural products with plant origin have many advantages such as abundant resources, low toxicity, diverse targets, and diverse molecular mechanisms. However, natural products not only have complex problems of extraction, isolation, purification and identification, but also poor solubility, poor permeability, low bioavailability, unstable biological environment, extensive metabolism, etc. In addition, some natural plants may have drug-drug interactions with chemotherapeutic drugs and effects of the body. It is necessary to find some delivery strategies that attempt to overcome these limitations and rediscover the new benefits associated with these natural products.
4 The paper does not discuss any limitations in terms of the generalizability of the findings to different types of drug-resistant cancers.
R: Thank you for your positive suggestion. This paper has discussed any limitations in the generalizability of the findings to different types of drug-resistant cancers. The modifications are as follows(see online 1030 to 1061 in paper）:
Studies have shown that different drug resistance phenotypes are associated with multi-ple rather than single alterations in the cell membrane, cytoplasm and nucleus of cancer cells. In ABC transporter proteins (e.g. P-gp, ABCB5, MRP1-5, BCRP), oxidative stress (e.g. GSH, GST, GGT, SOD, CAT, ROS), apoptotic proteins (Bcl-2, Bcl-xL, Bax, IAP, IκB, cysteine asparagine), microtubule proteins, DNA topoisomerases, histone deacetylases, oncogenes and oncogenes (epidermal growth factor receptor, c-Cbl, TP53), heat shock proteins, and a number of other proteins that undergo significant changes. Available studies have shown that plant-derived natural products act in different ways, however, since the mechanisms of tumor resistance are associated with multiple signaling pathways, there is a need to look for a broader range of multi-targeted drugs or combination therapy with existing drugs, however, there is a lack of clarity on the main targets and mechanisms of action of combination therapy as well as of multi-targeted natural products, which limits target interaction-based structure optimization and further development.
5 The paper does not mention any limitations in terms of the translation of the research findings into clinical applications or the development of anti-resistant-cancer drugs.
R: Thank you for your positive suggestion. This paper has discussed the limitations in translating research results into clinical applications or developing anti-resistant cancer drugs. The modifications are as follows(see online 1093 to 1115 in paper）:
Currently, the mechanisms of tumor multidrug resistance are complex and the mechanisms are rarely explored and relatively understudied in depth. In addition, most studies on cancer drug resistance mechanisms have focused on the cellular level and experimental animals, and therefore the actual clinical significance of these mechanisms is unknown and has not yet been more substantiated. Many challenging factors limit the development of natural anticancer biomolecules as drug products. Firstly, the development of natural products is a complex and time-consuming process, such as extraction, purification, isolation and characterization, etc. Moreover, in addition to toxic side effects, reduced water solubility, decreased absorption, and lack of selectivity for targeting cancer cells are the main obstacles to the development of anticancer drugs with natural origin.
6 The discussion should be rather organized around arguments avoiding simply describing details without providing much meaning.
R: Thank you for your positive suggestion. We have modified the discussion by removing unnecessary parts and adding that there are limitations with respect to different types of drug-resistant cancers. The modifications are as follows(see online 1030 to 1061 in paper）:
Malignant cancer is one of the diseases with the highest mortality rate globally, which seriously endangers human health of the world residents, and the number of cases is increasing with each passing year. A major reason for treatment failure in cancer pa-tients is the resistance of primary or acquired to chemotherapeutic agents. We have de-scribed most effective drug-resistant mechanisms in this review, including intracellular and extracellular pathways. The intracellular pathway includes reducing drug accumu-lation and absorption, inactivating or altering drug targets, inhibiting the expression of apoptosis-related genes, and altering membrane lipids. Meanwhile, there are other ex-tracellular factors contribute to the development of drug resistance, including EMT, CSCs and tumor microenvironment. It is ineffective and toxic that using high doses of drugs to overcome drug resistance, therefore, there is an urgent need to develop drugs with a better safety profile and higher efficacy for drug resistance cancer treatment. In recent years, plant-derived natural products and their secondary metabolites possess characteristics of abundant products, low toxicity and side effects, diverse biologic activities, and high content of active ingredients. It has been considered as the most promising candidates for oncology therapies. Our study reviewed that natural products have significantly antitumor effects on cancer, which showed possible benefits in treating cancer patients through numerous mechanisms, such as regulation of MDR related genes, inhibition of PI3K/AKT signaling pathway, induction of autophagy, and regulation of cell cycle arrest. Studies have shown that different drug resistance phenotypes are associated with multiple rather than single alterations in the cell membrane, cytoplasm and nucleus of cancer cells. In ABC transporter proteins (e.g. P-gp, ABCB5, MRP1-5, BCRP), oxidative stress (e.g. GSH, GST, GGT, SOD, CAT, ROS), apoptotic proteins (Bcl-2, Bcl-xL, Bax, IAP, IκB, cysteine asparagine), microtubule proteins, DNA topoisomerases, histone deacetylases, oncogenes and oncogenes (epidermal growth factor receptor, c-Cbl, TP53), heat shock proteins, and a number of other proteins that undergo significant changes. Available studies have shown that plant-derived natural products act in different ways, however, since the mechanisms of tumor resistance are associated with multiple signaling pathways, there is a need to look for a broader range of multi-targeted drugs or combination therapy with existing drugs, however, there is a lack of clarity on the main targets and mechanisms of action of combination therapy as well as of multi-targeted natural products, which limits target interaction-based structure optimization and further development.
7 Alkaloids and Terpenoids in Overcoming Drug Resistance: Need to add this section with mechanistic figure.
R: Thank you for your positive suggestion. We have added a mechanism map for alkaloids and terpenoids resistance, The figure is shown in the revised paper (see Fig 2).
8 Polyphenols and Flavonoids as Reversal Agents: Need to add this section with mechanistic figure.
R: Thank you for your positive suggestion. We have added a mechanism map for polyphenols and flavonoids resistance, The figure is the revised paper (see Fig 3).
9 Synergistic Effects and Combination Therapies: Need to add this section.
R: Thank you for your positive suggestion. We have added to synergistic and combination therapies. The modifications are as follows(see online 960 to 1002 in paper）:
Currently, the development of anticancer drugs is the mainstay of cancer treatment. However, there are many challenges associated with the use of single-agent therapy, including the emergence of drug-related side effects and drug resistance [272]. As a result, there has been an increasing interest in the strategies of combination therapies, which attempt to capitalize on synergistic effects by combining multiple drugs to treat multiple targets, subgroups, or diseases simultaneously [273]. Combination therapy significantly improves effectiveness compared to the traditional single-drug, single-target treatment paradigm. It has been found that EGCG is able to sensitize chemotherapy-resistant cancer cells, and it can act synergistically with various anticancer drugs in cancer treatment, such as cisplatin, oxaliplatin, temozolomide, resveratrol, doxorubicin, vardenafil, curcumin, erlotinib and others [274,275,276,277,278,279,280,281]. In human ovarian cancer SKOV3 and OVCAR3 cells, EGCG enhances sensitivity to cisplatin by up-regulating copper transporter protein 1 (CTR1), leading to the accumulation of intracellular cisplatin and cisplatin-DNA adducts, and the combination of EGCG and cisplatin inhibits tumor growth in OVCAR3 xenograft mice [274]. In addition, the combination of low concentrations of EGCG and curcumin significantly inhibits cell and tumor growth in human non-small cell lung cancer (NSCLC) A549 and NCI-H460 cells as well as in A549 xenograft mice [275]. Similarly, the combination of dihydroartemisinin (DHA) and gemcitabine had a strong synergistic effect on the loss of mitochondrial membrane potential and induction of apoptosis in human non-small cell lung cancer (NSCLC) A549 cells [282]. DHA also enhances the anticancer activity of the chemotherapeutic drug cisplatin in cispla-tin-resistant ovarian cancer cells [283]. In addition, ursolic acid (UA) in combination with doxorubicin enhances cellular uptake of adriamycin and reverses multidrug resistance (MDR) in human breast cancer MCF-7/ADR cells [284]. Tanshinone IIA is an effective drug to inhibit DOX resistance in gastric cancer by inducing cell cycle blockade. Combination with DOX enhances apoptosis and triggers autophagic cell death, increases the expression of p53, Bax, and LC3BII, and decreases the expression of Bcl-2 and p62. [285,286]
In addition to individualized compounds, a number of herbal compound components have been shown to have anti-tumor functions, and some are already in clinical trials. Shengmai Injection is a traditional Chinese medicine injection processed using modern pharmaceutical technology, and its active ingredients mainly include ginsenoside, oli-gosaponin, and tretinoin. Studies have shown that Shengmai Injection has the efficacy of potentiating chemotherapy [287], and the use of Shengmai Injection in combination with chemotherapeutic drugs can suppress the growth of transplanted tumors of VCR-resistant gastric cancer cells SGC7901 in nude mice, increase the lethality of chemotherapeutic drugs on gastric cancer cells, and accelerate the apoptosis of tumor cells [288]. The main ingredients in Yiqi Jianpi Huaji Tang include Astragalus, Radix et Rhi-zoma Ginseng, Rhizoma Atractylodis Macrocephalae, Radix Paeoniae Alba, Radix Paeo-niae Alba, Citrus aurantium, Fructus Lycii, Pine Root, and Salvia divinorum. Some scholars have pointed out that the combination of Yichijianpi Huaji Tang combine with 5-Fu can increase apoptosis and block the cell cycle in S phase. Yiqi Jianpi Huaji Tang in-creased the sensitivity of SGC7901/VCR cells to chemotherapy by decreasing the expression of MDR1/P-gp, MRP, TUBB3, and STMN1 [289].
10 Preclinical and Clinical Studies: Need to add this section.
R: Thank you for your positive suggestion. We have added to preclinical and clinical studies. The modifications are as follows(see online 1005 to 1028 and references 151-153,174-177 in paper）:
Clinical trials can confirm or reveal a drug's action, adverse effects, and pharmacokinetics. Tetrandrine is an isoquinoline alkaloid, which has been shown to significantly reduce the expression of P-gp and LRP and attenuate MDR. Clinical studies have shown that patients have a better toleration of Tetrandrine and its use as an adjuvant to chemotherapy [290]. Derivatives of Tetrandrine are effective in reversing MDR by inhibiting P-gp transporter and ATPase activity. The mechanism of this action is related to the blockade of the MEK-ERK (mitogen-activated protein kinase-extracellular signal-regulated protein kinase) signaling pathway [291]. 7,3′,4′-trihydroxyisoflavone（THIF） is the major metabolite of daidzein. It was found that adriamycin combined with THIF had better clinical efficacy in cervical cancer. THIF negatively regulates MDR1 by controlling transcription factors and then generates new MDRs [292]. In vivo, β-elemene significantly enhanced the antitumor activity of DOX and increased Caspase-3 protein expression in nude mice bearing SGC7901/ADR xenografts [293,294]. Shengmai injection combined with chemo-therapy can limit the growth of transplanted tumors of VCR-resistant gastric cancer cells SGC7901 in nude mice, increase the lethality of chemotherapeutic drugs on gastric cancer cells, and accelerate tumor cell apoptosis [295]. Qian et al. demonstrated that berberine can synergistically enhance the inhibitory effect of doxorubicin on tumor cell proliferation in MCF-7/DoxFluc, and the optimal combination ratio was Ber/DOX = 2:1 through using a luciferase reporter assay system combined with the bioluminescence imaging technology. In addition, it can significantly downregulate the expression of P-gp/ABCB 1 and MRP 1/ ABCC1 in vivo, reduce the efflux of DOX, increase the uptake of DOX in tumor tissues, and improve the concentration and retention rate of DOX in tumor cells [296]
11 Identification of challenges and limitations associated with the development and clinical translation of plant-derived natural products for overcoming drug resistance in cancer: Need to add this section.
R: Thank you for your positive suggestion. We have added challenges and limitations to the development and clinical translation of plant-derived natural products that overcome cancer drug resistance. The modifications are as follows:
Malignant cancer is one of the diseases with the highest mortality rate globally, which seriously endangers human health of the world residents, and the number of cases is increasing with each passing year. In recent years, plant-derived natural products and their secondary metabolites possess characteristics of abundant products, low toxicity and side effects, diverse biologic activities, and high content of active ingredients. It has been considered as the most promising candidates for oncology therapies. However, due to the known problems , such as extraction difficulties, poor solubility, poor permeability, low bioavailability, unstable biological environment, and extensive metabolism in the drug delivery system of natural products, in addition, certain natural plants may have drug-drug interactions with chemotherapeutic drugs and effects of the body. It is necessary to find some delivery strategies to overcome come these limitations and rediscover the new benefits associated with these natural products. In the study of drug resistance mechanisms, most of the current studies have focused on the classical mechanisms of the MDR gene and the P-gp encoded by the MDR gene, while few studies have focused on non-classical mechanisms such as GST-π, Topo II, DNA damage repair and tumor stem cells. In contrast, these mechanisms are rarely ex-plored and relatively understudied in depth. In addition, most studies on cancer drug resistance mechanisms have focused on the cellular level and experimental animals. However, the mechanisms of tumor multidrug resistance are complex, and therefore the actual clinical significance of these mechanisms is unknown and has not yet been more substantiated. Therefore, there is an urgent need for the development and clinical application of plant-derived natural products.
12 Future Directions and Therapeutic Potential: Need to add this section.
R: Thank you for your positive suggestion. We have added additions for future directions and therapeutic potential. The modifications are as follows:
In recent years, China's high-throughput technology has made great progress in the field of biomedicine, using high-precision technology, combined with modern pharmacology, pharmacodynamics, molecular biology and other new technologies to study the molecular mechanisms of tumors. It is expected that in the future, standardized treatment protocols in oncology will allow for the use of multiple compounds of natural origin to improve clinical efficacy and reduce the side effects of anticancer therapies. We believe that with the further systematization and comprehensiveness of research on immunity or the molecular pathology of cancer, natural products of plant origin will be widely applied in clinical medicine.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
The authors took into account the recommendations regarding the improvement of the quality of the article and therefore I believe that it can be accepted for publication in this form.
Author Response
Thank you for your encouraging comment ! It is a great honor to have your approval of this paper. Thank you for your positive suggestion to improve this article.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for Authors
I am satisfied with the revisions. In advance to some point, the introduction and discussion still lacks a crucial understanding. These papers need to read and cite in reference section.
- Multidrug Resistance in Cancer: Understanding Molecular Mechanisms, Immunoprevention, and Therapeutic Approaches
- Berberine as a potential anticancer agent: A comprehensive review
- Natural small molecules in breast cancer treatment: understandings from a therapeutic viewpoint
Comments on the Quality of English Language
Minor revisions
Author Response
Dear Editors and Reviewers,
Thanks for your letter concerning our manuscript entitled “Research Progress of Plant-Derived Natural Products against Drug Resistant Cancer”. We tried our best to improve the manuscript and made some changes to the manuscript. These changes will not influence the content and framework of the paper. Once again, thank you very much for your comments and suggestions.
Reviewer(s)' Comments to Author:
Reviewer: 2
Comments to the Author
1 I am satisfied with the revisions. In advance to some point, the introduction and discussion still lacks a crucial understanding. These papers need to read and cite in reference section.
R: Thank you for your positive suggestion. We have added some points into the Introduction part and Conclusion in the revised manuscript. (see the Highlights in paper）:
Introduction：
We add some points, such as :Studies have shown that multiple factors, such as epigenetics, micro RNAs (miRNAs), and long-stranded noncoding RNAs (lncRNAs), contribute to the development of multidrug resistance (MDR) in cancer cells [14-17]. Therefore, it is increasingly important to understand the molecular mechanisms that lead to the development of drug resistance, which has now been elucidated for many cancers and allows the use of conventional chemotherapeutic anticancer agents to cause DNA damage to kill drug-sensitive cancer cells [18,19]. However, in order to overcome cancer cell resistance, it is necessary to identify drugs that can be delivered to specific molecular targets in order to improve the specificity and precision of treatment. some of the references are listed below：(see the Highlights in paper on line 49-57）
[14] Chen X, Lu P, Wang D, Yang S, Wu Y, Shen H-Y, et al. The Role of miRNAs in Drug Resistance and Prognosis of Breast Cancer Formalin-Fixed Paraffin-Embedded Tissues. Gene (2016) 595:221–6.
[15] Han J, Sun W, Liu R, Zhou Z, Zhang H, Chen X, et al. Plasma Exosomal miRNA Expression Profile as Oxaliplatin-Based Chemoresistant Biomarkers in Colorectal Adenocarcinoma. Front Oncol (2020) 10:1495.
[16] Chen Y, Liu L, Li J, Du Y, Wang J, Liu J. Effects of Long Noncoding RNA (Linc-VLDLR) Existing in Extracellular Vesicles on the Occurrence and Multidrug Resistance of Esophageal Cancer Cells. Pathol Res Pract (2019) 215:470–7.
[17] Jin K-T, Lu Z-B, Lv J-Q, Zhang J-G. The Role of Long Non-Coding RNAs in Mediating Chemoresistance by Modulating Autophagy in Cancer. RNA Biol (2020) 17:1727–40.
[18] Cheung-Ong K, Giaever G, Nislow C. DNA-Damaging Agents in Cancer Chemotherapy: Serendipity and Chemical Biology. Chem Biol (2013) 20:648–59.
[19] Mansoori B, Mohammadi A, Davudian S, Shirjang S, Baradaran B. The Different Mechanisms of Cancer Drug Resistance: A Brief Review. Adv Pharm Bull (2017) 7:339–48.
Conculsion:
We add some points, such as :In addition, genetic mutations, epigenetic alterations including DNA methylation, histone alterations, and miRNAs can modulate the development of multidrug resistance [303]. (see the Highlights in paper on line 1049-1051）
We add some points, such as :However, due to the lack of therapeutic targets and therapeutic strategies, more research on natural products is needed to characterize their mechanisms of action and possible roles in MDR therapy. Researchers have now explored various methods and approaches to overcome drug resistance. Particles, nanomedicines and gene editing techniques such as CRISPR/Cas9 have been discovered to overcome multidrug resistance in tumor cells [303]. (see the Highlights in paper on line 1071-1076）
[303] Bin T E ,Asif S ,Rafi A M , et al.Multidrug Resistance in Cancer: Understanding Molecular Mechanisms, Immunoprevention and Therapeutic Approaches#13;[J].Frontiers in Oncology,2022,12891652-891652.
We add some points, such as :Resistance may arise due to alterations in the stroma and tumor microenvironment, and anticancer drugs may generate resistance by altering the target, drug efflux pumps, increasing cell tolerance to apoptosis, and accelerating tumor cell proliferation [304]. some of the references are listed below：(see the Highlights in paper on line 1094-1096）
[304] Abdur R ,Tareq A ,Ahmed A K , et al.Berberine as a Potential Anticancer Agent: A Comprehensive Re-view[J].Molecules,2021,26(23):7368-7368.
We add some points, such as :Two methods of prevention have been identified in the fight against cancer. One is chemo-prevention and the other is immunoprevention [305]. Currently the best method is considered to be immunoprophylaxis. However its potential side effects, toxicity, mutations, and immune checkpoint modulation remain significant issues and limitations that prevent this method from reaching clinical application [306]. some of the references are listed below：(see the Highlights in paper on line 1117-1121）
[305] Gu KJ, Li G. An Overview of Cancer Prevention: Chemoprevention and Immunoprevention. J Cancer Prev (2020) 25:127
[306] Umar A. Cancer Immunoprevention: A New Approach to Intercept Cancer Early. Cancer Prev Res (2014) 7:1067–71.
Author Response File: Author Response.docx
