Effects of plumbagin analogs on the JAK-STAT pathway

An Honors Thesis

Presented by

William Dean Lynch III

Completion Date:
May 2022

Approved By:

_____________________________________________
Dr. Richard Schur, Honors Director

_____________________________________________
Dr. Madhuri Manpadi, Chemistry Department

Drury University, 2022

© 2022, William Dean Lynch III
 Lynch 2

ABSTRACT
Title: Effects of Plumbagin Analogs on the JAK-STAT Pathway
Author: William Dean Lynch III
Thesis/Project Type: Project

This research studies the effect of plumbagin analogs on the JAK-STAT pathway.
Plumbagin is a promising anticancer compound and a known inhibitor of this pathway.
Plumbagin however lacks bioavailability and is not selective for cancer when administered
intravenously. This has led to the exploration of plumbagin analogs that aim to improve upon
plumbagin's poor bioavailability. The inhibition of the JAK-STAT pathway by analogs of
plumbagin were tested on U266 cells via western blotting. Results showed that the chosen
compound did not inhibit this pathway as much as plumbagin.
 Lynch 3

Effects of Plumbagin Analogs on the JAK-STAT Pathway

William Dean Lynch III

Abstract

This research studies the effect of plumbagin analogs on the JAK-STAT pathway.

Plumbagin is a promising anticancer compound and a known inhibitor of this pathway.

Plumbagin however lacks bioavailability and is not selective for cancer when administered

intravenously. This has led to the exploration of plumbagin analogs that aim to improve upon

plumbagin's poor bioavailability. The inhibition of the JAK-STAT pathway by analogs of

plumbagin were tested on U266 cells via western blotting. Results showed that the chosen

compound did not inhibit this pathway as much as plumbagin.

Introduction

The pursuit of anticancer pharmaceuticals has been around ever since the disease they

aim to treat has been characterized. Out of this pursuit, the first attempts at therapeutic

compounds were derived from folk-medicine. Naturally occurring substances were among the

first to be tested and studied1. One naturally occurring compound that has begun testing is

5-hydroxy-2-methyl-1,4-naphthoquinone, better known as plumbagin.

Plumbagin, pictured in fig. 1, has been shown to have numerous

anticancer properties10. It was determined to exert anticancer properties

on breast cancer2, lung cancer3, 4, ovarian cancer5, acute promyelocytic

leukemia6, melanoma7, prostate cancer8, 9. Santosh, et. al. characterized

the anticancer properties of plumbagin and found that plumbagin as a molecule has anticancer

properties, mainly through modulation of the STAT3 pathway10 (fig. 2). Signal transducers and
 Lynch 4

activators of transcription 3, STAT3, is

an important complex, which is

important for carcinogenesis,

proliferation, and angiogenesis10.

Plumbagin, by binding to a protein

tyrosine phosphatase, SHP-1, causes a

conformational change in the STAT3

protein that leads to inhibition of c-Src,

JAK-1, and JAK-2 activation, and may

make cancers which overexpress STAT3 to become more sensitive to apoptosis. Plumbagin has

been found to upregulate the tumor-suppressor genes p53 and p51, resulting in the cell to be

arrested in G2/M phase4. This is likely due to the overexpression of the STAT3 pathway, showing

that plumbagin might be a promising therapeutic agent.

Despite the promising outlook of plumbagin as treatment for a wide range of cancers, it is

neglected to be used today due to poor bioavailability. Aziz et. al. was able to find that

plumbagin selective targeted inhibited the growth and development of prostate cancer tumors,

while leaving prostate epithelial cells less vulnerable to the apoptotic effects of plumbagin3, but

other sources found conflicting evidence. The general concept of plumbagin is that once it is

administered intravenously, it fails to selectively reach and target tumors18. Further, it fails to

affect secondary tissue as well. Plumbagin's inability to selectively target tumors has led to its

lack of use in treating cancer.

A pivotal component of medicinal chemistry is developing and identifying structural

analogs to functional molecules in effort to improve upon potency and functionality—a process
 Lynch 5

termed combinatorial chemistry. From

this idea, plumbagin analogs have often

been synthesized and researched13. The

lab of Dr. Manpadi has previously

synthesized several analogs of

plumbagin, which were used

experimentally (fig. 3). Ongoing studies

to understand the mechanism of action of these analogs

are being taken—the purpose of this research is to test the efficacy of plumbagin analogs thus

further characterizing the anticancer properties of plumbagin, and identifying functional analogs

for future research.

Materials and Methods

U266 cells, human B-cells extracted from a patient with Multiple Myeloma, were

obtained from ATCC. For this research, it was important to have a cell line with the STAT3

Pathway upregulated. One way in which this pathway is mediated is through interleukin-6. For

this research, the U266 cell line was chosen since they upregulate human IL-611. Thus, the

STAT3 pathway is expected to be upregulated in the selected cell line. This allows the inhibition

by our plumbagin analogs more observable in U266 cell lines. The cell line was grown in RPMI

media, (84% RPMI, 15% FBS, 1% Penicillin/Streptomycin). Cells were split, and media was

renewed every 3-4 days. Cells were grown until their concentration was viable for testing. Cell

concentration was determined by use of a C-chip hemocytometer. 1 mL of 2 million cells/mL

was plated in each well in a 16 well plate. The wells were then incubated at 37 ºC with DMSO as
 Lynch 6

a negative control, Plumbagin as a positive control, and the

plumbagin analogs to be tested at a concentration of 25 µM.

After incubation for 5 hours, in order to isolate the

proteins, cells were to be lysed. Cell lysing began by transferring

the 1 mL of treated cells into an eppendorf, followed by

centrifugation, and the supernatant removed. The pellet was then

broken, and to each eppendorf, 70 µL of RIPA lysis buffer was

added, and set on ice for 30 minutes, sonicated three times at 0, 15,

and 30 minutes. Then cells were then centrifuged again, and the

supernatant was extracted in order to obtain the protein lysate. The

protein lysate then transferred to new, labeled eppendorfs and

frozen to be used in further analysis.

SDS-PAGE

The proteins obtained via cell lysing were separated using

a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A general idea of

SDS-Page is provided in fig. 4. The previously obtained frozen protein lysates were obtained and

thawed on ice, along with 1 mL of 5x sample buffer with 1µL Dithiothreitol (DTT), and protein

ladder. Once thawed, 12 µL of 5x sample buffer and DTT was added to each sample, then boiled

for 5 minutes at 90-100 ºC and centrifuged for 5 minutes. Following, the polyacrylamide gel, 1x

running buffer, and an electrophoresis chamber were obtained. The gel was placed in the

electrophoresis chamber and subsequently filled with 1x running buffer. Each of the 15 wells

were then filled with the protein lysates in a manner similar to fig. 5. 10 µL of Ladder was added

to wells designated L, and 15 µL of lysate was designated to wells of D (DMSO at 25 µM), P

 Lynch 7

(plumbagin at 25 µM), and C (compound at 25 µM). The wells were assigned in this way so that

the proteins could be transferred to the nitrocellulose membrane and tested at differing antibody

concentrations.

The SDS-PAGE was then run at 130v for roughly an hour, or until the blue line, a

bromophenol dye from the loading buffer, was near the bottom of the gel. The purpose of

SDS-PAGE is to separate proteins by molecular weight. Sodium Dodecyl Sulfate binds

nonspecifically to each protein, giving them a uniform negative charge. Then, running the gel at

130v will move all the charged proteins to the anode. The proteins with less molecular weight

will travel faster towards the bottom of the gel than heavier proteins.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

L D P C L D P C L D P C D P C
Figure 5

Western Blotting

Western blotting begins with the finished gel

obtained by the SDS-PAGE procedure, shown in

figure 6. First, the proteins separated via this method

were to be transferred to a nitrocellulose membrane.

A gel, nitrocellulose membrane, filter papers,

sponges, and a sandwich were obtained. The

sandwich was then assembled similar to as

depicted in fig. 7. The purpose of transferring to

the nitrocellulose membrane is to move the

proteins to a medium (in this case the membrane)

 Lynch 8

to a surface where they can be treated more easily, as

well as a membrane that can be cut so that another

variable can be measured.

Once the sandwich is assembled, it is run in the

electrophoresis chamber for two hours at 65v. The

proteins then again follow the charge and transfer to

the nitrocellulose membrane from the gel. In order to

test if the proteins did in fact transfer, ponceau S was used to stain the blot as seen in fig. 8.

Using the stained membrane, scissors can be used to cut the membrane into strips. The divisions

can be made as shown in fig. 5.

With the nitrocellulose membrane divided into strips, antibody treatment at various

dilutions were started. For initial studies, Phosphorylated JAK-2 antibodies isolated from rabbit

were decided to be studied. First, the proteins in the membranes were blocked with 5% Bovine

Serum Albumin in TBST for 1.5 hours. The purpose of blocking is to prevent non-specific

interactions between the antibody treatment and any undesired proteins. After blocking, the

membranes were treated with primary Phosphorylated JAK-2 antibodies at varying

concentrations. The concentrations of primary antibodies tested experimentally were 1:1000,

1:5000, 1:10,000, and 1:20,000. The membranes were then incubated overnight at 4 ºC on a

shaker. The next day, secondary antibody treatment would occur, which would bind to the

attached P JAK-2 antibodies. Secondary antibodies were left to incubate at a concentration of

1:5000 for 3 hours at room temperature. After secondary antibody treatment, a colorimetric dye

was used to visualize the blot, providing observable results.

 Lynch 9

Results

After several trials, and optimization of the procedure, bands were ultimately obtained

and significant results could be observed. These results will be described below. Cells were

counted and a 1 mL of a concentration of 2 x 106 cells/mL was plated into each well. Six wells

were then incubated for five hours. Two wells each were incubated with DMSO, plumbagin, and

compound 11 (seen in fig. 3).

For SDS-PAGE, and Western Blotting, fig. 9 shows how the compounds were assigned to

the following wells, and the corresponding antibody concentrations were used.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

L D P 11 L D P 11 L D P 11 D P 11

1 : 1,000 1 : 5,000 1 : 10,000 1 : 20,000

1 : 5,000 1 : 5,000 1 : 5,000 1 : 5,000

Figure 9

Figure 10
 Lynch 10

Following treatment by colorimetric dye, these results yielded the presence of bands on

the nitrocellulose membrane, pictured in Figure 10. Results of Western Blotting indicate that the

control of DMSO did not inhibit proteins at around 150 - 100 KD. Plumbagin was able to

successfully inhibit proteins at this mark, whereas the compound tested, compound 11, did not

inhibit these proteins.

Discussion

The results indicate that the JAK-STAT pathway was not inhibited by the chosen

compound. Using DMSO as the control, it can be seen that there was less inhibition of the

P-JAK protein than in that of plumbagin in every concentration of antibody. Compound 11

showed similar inhibition to that of DMSO, indicating that it was not successful in inhibiting the

JAK-STAT pathway. Plumbagin and compound 11 are depicted in Figure 11, and their Structure

Activity Relationships will be analyzed.

Both plumbagin and compound 11 are 1,4-naphthoquinones. In many signaling pathways,

plumbagin can disrupt the possible anti-apoptotic signals of many cancers through direct binding

with the partial negative charges generated at the 1 and 4 carbons[31]. Thus, this structure was

preserved in compound 11. Compound 11 differs from plumbagin at C-5 and C-2. At carbon-5,

compound 11 retains a hydrogen, whereas plumbagin has an alcohol at this position. The

presence of this alcohol often aids to stabilize plumbagin in its binding pocket[31], meaning that
 Lynch 11

compound 11’s apparent lack of inhibition may have come from this modification. The second

modification found in compound 11 was at the carbon-2 position; as seen in figure 11, this is a

large structural change. This addition greatly modifies the molecular weight of the molecule,

possibly adds steric hindrance to the binding pocket, and adds both an aromatic ring, and polar

nitrogens to the molecule. The intention behind this addition appears to remedy the problem of

plumbagin that it cannot selectively target cancer cells once administered intravenously.

Experimentally however, compound 11 lacks the same ability to inhibit the JAK-STAT.

This leads to the conclusion that the bulky addition to the carbon-2 position does not result in

similar levels of inhibition of P-JAK 2 as plumbagin, and further testing on compound 11 is

suggested at this time.

After several trials, and optimization of the procedure, bands were ultimately obtained

and significant results could be observed. Many early trials did not yield observable results; the

western blots lacked the presence of bands where the P-JAK 2 proteins should have been present.

This could have been due to using too few cell antibodies that may have been inactive. In the

final trial of the procedure, new concentrations of both primary and secondary antibodies were

prepared from a fresh stock of antibodies, and different dilutions were tested. From creating new

concentrations of antibodies from the stock solutions, observable results were obtained,

indicating that this modification helped deliver results..

Many other modifications to the procedure occurred during the course of the experiment

in effort to develop a method that is viable. A second modification made was the amount of cell

lysate added to each well during SDS-PAGE. This number was changed from 12 µL to 15 µL.

This was done in effort to increase the density of protein in the well, so that eventual bands

would appear darker on the western blot. Another modification made was during each incubation
 Lynch 12

of the experiment. Cells were often incubated with the compounds for times longer than the

originally proposed four hours, yet the final incubation time was five hours. During incubation

with primary antibody, we instead incubated overnight at 4 ºC instead of incubating at room

temperature for 1.5 hours. This was done so that the antibody might better bind to the intended

proteins. The western blot was also incubated with a secondary antibody for slightly longer, for a

time of 3 hours rather than 2-2.5 so that the secondary antibody might better attach to the

primary.

Altogether, these modifications combined to observable and meaningful results. The two

of the most significant changes exist in preparation of fresh primary antibodies, and incubation

of these primary antibodies. In future experiments, the modification to the incubation of primary

antibodies is most vital to preserve.

During research, limitations existed. These included inadequate time, occasional lack of

materials/inability to find the materials, contamination, and others. The greatest of these

limitations was inadequate time. Future research is recommended, as there are many more

compounds to test. Additionally, one successful western blot of compound 11 is not sufficient

evidence to effectively rule out this compound as a viable analog of plumbagin. Compound 11

may have anticancer effects on U266 cells at concentrations other than plumbagin, or it may

exert its effects on other pathways also inhibited by plumbagin.

For future research, prepare fresh aliquots of antibodies at new concentrations. Out of the

concentrations of primary antibodies tested, the concentration that showed the most visible band

was the 1:20,000 concentration, indicating that there might be a more diluted concentration that

could yield better results. Bands however were still not as clear as desired; different antibody

concentrations are recommended for testing so that a more clear western blot can be obtained.
 Lynch 13

Incubation with primary antibody was greatly manipulated experimentally and this change

improved the quality of the results. This modification is recommended in successive

experiments. Additionally, future research should continue to test other compounds from the

library available in Manpadi lab. Differing times of incubation of these compounds may also be a

variable that can be changed to yield better results, as cells were incubated with compounds at a

range of time across experiments.

In conclusion, the compound 11 did not show the same levels of inhibition of the

JAK-STAT pathway as plumbagin. There is not sufficient evidence to support this claim

however. Compound 11 may be more efficacious at different concentrations than that of

plumbagin or it may inhibit another pathway, delivering anticancer effects. Other conditions

should be tried for proper compound analysis. The antibody concentrations may not have been

ideal, as the bands were not as clear as desired. This research cannot be concluded as final as

there is a wide breadth of research still able to be done. Future research is recommended to

confirm these findings.

Acknowledgements

I thank Dr. Madhuri Manpadi at Drury University for her patience, guidance, and

contributions over the course of the research; I thank Dr. Richard Schur for his support and

guidance; I thank Morgan Wallace and Zina Umran for their assistance during this project; I

thank Drury University for the opportunity to do purposeful and meaningful research in a field

that greatly interests me; And finally, I thank everybody who has encouraged and guided me

along the way.

 Lynch 14

Literature Cited

[1] Alem, F. Z., Bejaoui, M., Villareal, M. O. , Rhourri-Frih, B., & Isoda, H. (2020). Elucidation
of the effect of plumbagin on the metastatic potential of B16F10 murine melanoma cells via
MAPK signalling pathway. Exp Dermatol, 29(4), 427-435. DOI: 10.1111/exd.14079.

[2] ATCC. (2021). U266B1 [U266]. https://www.atcc.org/products/tib-196.

[3] Aziz, M. H., Dreckschmidt, N. E., & Verma, A. K. (2008). Plumbagin, a medicinal
plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone
refractory prostate cancer. Cancer Res, 68(21), 9024-32. DOI:
10.1158/0008-5472.CAN-08-2494.

[4] Gomathinayagam, R., Sowmyalakshmi, S., Mardhatillah, F., Kumar, R., Akbarsha, M. A., &
Damodaran, C. (2008). Anticancer mechanism of plumbagin, a natural compound, on non-small
cell lung cancer cells. Anticancer Res, 28(2A), 785-92. PMID: 18507021.

[5] Hsu, Y. L., Cho, C. Y., Kuo, P. L., Huang, Y. T., & Lin, C. C. (2006). Plumbagin (5-hydroxy-
2-methyl-1,4- naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53
accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and
in vivo. J Pharmacol Exp Ther, 318(2), 484-94. DOI: doi: 10.1124/jpet.105.098863.

[6] Kuo, P. L., Hsu, Y. L., & Cho, C.Y. (2006). Plumbagin induces G2-M arrest and autophagy
by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol
Cancer Ther, 5(12), 3209-21. DOI: 10.1158/1535-7163.MCT-06-0478.

[7] Kijewski, A., Witsø, I. L., Iversen, H., Rønning, H. T., L'Abée-Lund, T., Wasteson, Y.,
Lindbäck, T., & Aspholm, M. (2020). Vitamin K Analogs Influence the Growth and Virulence
Potential of Enterohemorrhagic Escherichia coli. Appl Environ Microbiol, 86(24), 583-29. DOI:
10.1128/AEM.00583-20.

[8] Latosińska, J. N., & Latosińska, M. (2013). Anticancer drug discovery - from Serendipity to
rational design. IntechOpen. DOI: 10.5772/52507.

[9] Mahmood, T., & Yang, P. C. (2021). Western Blot: Technique, Theory, and Troubleshooting.
N Am J Med Sci, 4(9), 429-34. DOI: 10.4103/1947-2714.100998.

[10] Matsushime, H., Roussel, M. F., Ashmun, R. A., & Sherr, C. J. (1991). Colony-stimulating
factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell, 65(4), 701-13. DOI:
10.1016/0092-8674(91)90101-4.
 Lynch 15

[11] National Center for Biotechnology Information. (2021). CCND1 cyclin D1 [ homo sapiens (
human ) ]. Gene ID: 595. https://www.ncbi.nlm.nih.gov/gene/595.

[12] Nayak, N., Bajpai, M., & Razdan, B. (2014). Plumbagin analogs-synthesis, characterization,
and
antitubercular activity. J Adv Pharm Technol Res, 5(1), 28-32. DOI: 10.4103/2231-4040.126984.

[13] Padhye, S., Dandawate, P., Yusufi, M., Ahmad, A., & Sarkar, F. H. (2010). Perspectives on
medicinal properties of plumbagin and its analogs. Med Res Rev, 36(6), 1131-58. DOI:
10.1002/med.20235.

[14] Panichayupakaranant, P., & Ahmad, M. I. (2016). Plumbagin and its Role in Chronic
Diseases. Adv Exp Med Biol, 2016(929), 229-46. DOI: 10.1007/978-3-319-41342-6_10.

[15] Pereyra, C. E., Dantas, R. F., Ferreira, S. B., Gomes, L. P., & Silva-Jr, F. P. (2019). The
diverse mechanisms and anticancer potential of naphthoquinones. Cancer Cell Int, 19, 207. DOI:
10.1186/s12935-019-0925-8.

[16] Powolny, A. A., & Singh, S. V. (2008). Plumbagin-induced apoptosis in human prostate
cancer cells is associated with modulation of cellular redox status and generation of reactive
oxygen species. Pharm Res, 25(9), 2171-80. DOI: 10.1007/s11095-008-9533-3.

[17] Rajalakshmi, S., Vyawahare, N., Pawar, A., Mahaparale, P., & Chellampillai, B. (2018).
Current development in novel drug delivery systems of bioactive molecule plumbagin. Arti Cells
Nanomed Biotechnol, 46(1), 209-218, DOI: 10.1080/21691401.2017.1417865.

[18] Sakpakdeejaroen, I., Somani, S., Laskar, P., Irving, C., Mullin, M., & Dufès, C. (2020).
Anti-Tumor Activity of Intravenously Administered Plumbagin Entrapped in Targeted
Nanoparticles. J Biomed Nanotechnol, 16(1), 85-100. DOI: 10.1166/jbn.2020.2874.

[19] Sandur, S. K., Pandey, M. K., Sung, B., & Aggarwal BB. (2010). 5-hydroxy-2-methyl-1,4-
naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through
induction of protein tyrosine phosphatase, SHP-1: potential role in chemosensitization. Mol
Cancer Res, 8(1), 107-18. DOI: 10.1158/1541-7786.MCR-09-0257.

[20] Shi, Y. (2004). Caspase activation, inhibition, and reactivation: A mechanistic view. Protein
Sci, 13(8), 1979-87. DOI: 10.1110/ps.04789804.
 Lynch 16

[21] Srinivas, P., Gopinath, G., Banerji, A., Dinakar, A., Srinivas, G. (2004). Plumbagin induces
reactive oxygen species, which mediate apoptosis in human cervical cancer cells. Mol Carcinog,
40(4), 201-11. DOI: 10.1002/mc.20031.

[22] Sung, B., Oyajobi, B., & Aggarwal, B. B. (2011). Plumbagin Inhibits Osteoclastogenesis
and Reduces Human Breast Cancer-Induced Osteolytic Bone Metastasis in Mice through
Suppression of RANKL Signaling. Mol Cancer Ther, 11(2), 250-9. DOI:
10.1158/1535-7163.MCT-11-0731.

[23] Thasni, K. A., Rakesh, S., Rojini, G., Ratheeshkumar, T., Srinivas, G., & Priya, S. (2008).
Estrogen- dependent cell signaling and apoptosis in BRCA1-blocked BG1 ovarian cancer cells in
response to plumbagin and other chemotherapeutic agents. Ann Oncol, 19(4), 696-705. DOI:
10.1093/annonc/mdm557.

[24] Thasni, K. A., Ratheeshkumar, T., Rojini, G. Sivakumar, K. C., Rakesh, S. N., Srinivas, G.,
Banerji, A., Somasundaram, V., & Srinivas, P. (2013). Structure activity relationship of
plumbagin in BRCA1 related cancer cells. Mol. Carcinog, 52(5), 392-403. DOI:
10.1002/mc.21877.

[25] The European Bioinformatics Institute. (2021). CHEBI:8273 - plumbagin.

https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:8273.

[26] Wang, C. C., Chiang, Y. M., Sung, S. C., Hsu, Y. L., Chang, J. K., & Kuo, P. L. (2008).
Plumbagin induces cell cycle arrest and apoptosis through reactive oxygen species/c-Jun
N-terminal kinase pathways in human melanoma A375.S2 cells. Cancer Lett, 259(1), 82-98.
DOI: 10.1016/j.canlet.2007.10.005.

[27] Wang, S. W., & Sun, Y. M. (2014). The IL-6/JAK/STAT3 pathway: Potential therapeutic
strategies in treating colorectal cancer (Review). Int J Oncol, 44(4), 1032-30. DOI:
10.3892/ijo.2014.2259.

[28] Wu, H., Dai, X., & Wang, E. (2016). Plumbagin inhibits cell proliferation and promotes
apoptosis in multiple myeloma cells through inhibition of the PI3K/Akt-mTOR pathway. Oncol
Lett, 12(5), 3614-18. DOI: 10.3892/ol.2016.5048.

[29] Yin, Z., Zhang, J., Chen, L., Guo, Q., Yang, B., Zhang, W., & Kang, W. (2020). Biomed Res
Int, 2020, 6940953. DOI: 10.1155/2020/6940953.

[30] Zhao, Y. L., & Lu, D. P. (2006). Effects of plumbagin on the human acute promyelocytic
leukemia cells in vitro. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 14(2), 208-11). PMID: 16638181.
 Lynch 17

[31] Jamal, M. S., Parveen, S., Beg, M. A., Suhail, M., Chaudhary, A. G., Damanhouri, G. A.,
Abuzenadah,. A. M., & Rehan, M. (2014). Anticancer compound plumbagin and its molecular
targets: a structural insight into the inhibitory mechanisms using computational approaches. PloS
one 9(2), 87309. PMID: 24586269.