reactive oxygen species (ROS); TrxR1; CRM1; PI3K/Akt/mTOR
Piperlongumine (PPLGM) is described as a reactive oxygen species (ROS) inducer. Its anticancer effects are mediated through ROS-induced DNA damage.[1]

Piperlongumine is a known ROS inducer that has the potential to cause the death of pancreatic cancer cells in culture[1] Platelet aggregation is prevented by piperlongumine, a thromboxane A(2) receptor antagonist. [2] In addition to promoting autophagy and mediating cancer cell death, piperlongumine inhibits Akt/mTOR signalling.[3]

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Piperlongumine (PPLGM) inhibited the growth of three human pancreatic cancer cell lines (PANC-1, MIA PaCa-2, and BxPC-3) in a concentration- and time-dependent manner, with IC50 values of 4.2 µM, 4.6 µM, and 4.2 µM, respectively, after 72 hours of treatment, as measured by alamarBlue assay.[1]
Piperlongumine (PPLGM) reduced the long-term clonogenic survival of pancreatic cancer cells in a concentration-dependent manner. Treatment with 5 µM and 10 µM PPLGM completely abolished colony formation in MIA PaCa-2 and BxPC-3 cells.[1]
Piperlongumine (PPLGM) (10 µM) significantly elevated intracellular ROS levels after 6 hours of treatment in all three pancreatic cancer cell lines, as measured by DCF-DA assay and flow cytometry. The fold increases in ROS relative to control were approximately 9-fold for PANC-1, 24-fold for MIA PaCa-2, and 8-fold for BxPC-3.[1]
Treatment with Piperlongumine (PPLGM) (10 µM) for 24 hours induced DNA fragmentation (DNA laddering) in MIA PaCa-2 cells, similar to the positive control gemcitabine (5 µM). This DNA damage was quantified by densitometry.[1]
Western blot analysis showed that Piperlongumine (PPLGM) (10 µM) treatment for 24 hours increased the phosphorylation of Chk1 at Ser296 (pChk1 S296) in MIA PaCa-2 cells, indicating activation of the DNA damage response.[1]
Co-treatment with the antioxidant N-acetyl cysteine (NAC, 3 mM) partially reversed Piperlongumine (PPLGM)-induced cell death and reduced DNA fragmentation in MIA PaCa-2 cells, suggesting that ROS generation is central to its cytotoxic mechanism.[1]
Quantitative PCR analysis showed that Piperlongumine (PPLGM) (10 µM) treatment for 24 hours did not significantly alter the mRNA expression levels of key antioxidant enzymes (SOD1, GSTP1, HO1) in PANC-1 cells.[1]

Piperlongumine (50 mg/kg i.p.) causes tumor cell in vivo growth inhibition without significantly altering biochemical, hematological, or histopathological parameters. [4]

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In a PANC-1 xenograft mouse model, daily intraperitoneal administration of Piperlongumine (PPLGM) at 2.4 mg/kg for 30 days significantly suppressed tumor growth compared to the vehicle control (1% DMSO). The final tumor mass in the treated group was approximately 50% lower than in the control group.[1]
Immunohistochemical analysis of tumors from treated mice revealed a significant decrease in the proliferation marker Ki-67 and a significant increase in the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), compared to tumors from control mice.[1]
The treatment was well-tolerated, with no significant differences observed in final body weight or the weights of major organs (kidney, liver, lung, spleen) between the treated and control groups.[1]

Various PL concentrations are incubated with MCF-7 and 786-O cells for 48 hours. CellTiter Blue assay is used to analyze cell proliferation. With the help of the Microsoft Excel add-in XLift, effective doses (ED) are calculated.

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AlamarBlue Cell Viability Assay: PANC-1, MIA PaCa-2, and BxPC-3 cells were seeded in 96-well plates (5.0×10³ cells/well). After 24 hours, cells were treated with varying concentrations of Piperlongumine (PPLGM) (0–20 µM). AlamarBlue reagent was added to a final concentration of 10% and incubated for 4 hours at 37°C. Absorbance was measured at 570 nm and 600 nm. Percent reduction of alamarBlue was calculated to determine cell viability relative to control.[1]
Clonogenic Survival Assay: PANC-1, MIA PaCa-2, and BxPC-3 cells were seeded in 24-well plates at a low density (5×10² cells/well). The next day, cells were treated with varying concentrations of Piperlongumine (PPLGM) (0–20 µM) for 24 hours. The drug-containing medium was then replaced with fresh medium, and cells were allowed to grow undisturbed for 14 days to form colonies. Colonies were fixed, stained with crystal violet, and manually counted.[1]
Intracellular ROS Measurement (DCF-DA Assay): Pancreatic cancer cells (5.0×10⁵ cells/mL) were treated with 10 µM Piperlongumine (PPLGM) for 6 hours. Cells were then harvested, resuspended in PBS containing 10 µM DCF-DA, and incubated at 37°C for 30 minutes. The fluorescence intensity of oxidized DCF, indicative of ROS levels, was measured by flow cytometry.[1]
DNA Fragmentation Assay (DNA Laddering): MIA PaCa-2 cells (5.0×10⁵ cells) were treated with 10 µM Piperlongumine (PPLGM), 5 µM gemcitabine (positive control), or vehicle for 24 hours. Genomic DNA was extracted, resolved on a 1.5% agarose gel containing ethidium bromide, and visualized under UV light to assess DNA fragmentation.[1]
Western Blot Analysis: MIA PaCa-2 cells (1×10⁶ cells) were treated with 10 µM Piperlongumine (PPLGM) or 5 µM gemcitabine for 24 hours. Cells were lysed, and proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies against phosphorylated Chk1 (S296) and total Chk1. Protein bands were detected using chemiluminescence.[1]
Quantitative PCR (qPCR): Total RNA was extracted from PANC-1 cells treated with or without 10 µM Piperlongumine (PPLGM) for 24 hours. cDNA was synthesized, and mRNA levels of antioxidant genes (SOD1, GSTP1, HO1) were quantified using SYBR Green-based real-time PCR, with 18S rRNA as an internal control.[1]

Mice transplanted with sarcoma 180 tumors
~50 mg/kg
i.p.

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Xenograft Model and Treatment: Female athymic nude mice (6–8 weeks old) were subcutaneously injected with 2.0×10⁶ PANC-1 cells in the rear flank. Once palpable tumors formed, mice were randomly divided into two groups (n=8). The control group received daily intraperitoneal injections of vehicle (1% DMSO in PBS). The treatment group received daily intraperitoneal injections of Piperlongumine (PPLGM) at 2.4 mg/kg body weight (dissolved in DMSO and further diluted in PBS to a final DMSO concentration of 1%) for 30 consecutive days. Tumor dimensions were measured three times per week, and tumor volume was calculated using the formula V = (L × W²)/2. Body weight was monitored weekly. After 30 days, mice were euthanized, and tumors and major organs were harvested for analysis.[1]

In xenograft mice, daily intraperitoneal injection of Piplartine (PPLGM) (2.4 mg/kg) for 30 days was well tolerated. There were no significant differences in final body weight, kidney, liver, lung, and spleen weight between the treatment and control groups, indicating that no significant systemic toxicity was observed at this dose and administration regimen. [1]

[1]. Toxicol Rep . 2014:1:309-318.

[2]. Eur J Pharmacol . 2007 Sep 10;570(1-3):38-42.

[3]. Nat Prod Res . 2014;28(22):2040-3.

[4]. J Appl Toxicol . 2008 Jul;28(5):599-607.

[5]. Cancer Lett . 2016 May 28;375(1):114-126.

[6]. Chem Biol Interact . 2015 Jul 25:237:66-72.

[7]. Apoptosis . 2014 Jul;19(7):1148-64.

Piplartine belongs to the cinnamamide class of compounds and is a dicarboxyimide. Piplartine has been reported to exist in Piper arborescens, Piper puberulum, and other organisms with relevant data. See also: Piper arborescens (partial). Piplartine (PPLGM) is a bioactive alkaloid derived from the Piper arborescens plant. It has been reported to selectively induce cancer cell death by increasing reactive oxygen species (ROS) levels, with no significant effect on normal cells. This study confirmed its effectiveness regardless of the K-ras mutation status of pancreatic cancer cells (PANC-1 and MIA PaCa-2 are mutant, BxPC-3 is wild-type). Its mechanism of action involves ROS-induced DNA damage, which in turn activates DNA damage responses (such as Chk1 phosphorylation), ultimately leading to cell death. These findings support its potential as a therapeutic agent for pancreatic cancer. [1]

C17H19NO5

317.3365

317.126

C, 64.34; H, 6.03; N, 4.41; O, 25.21

20069-09-4

20069-09-4

637858

White to yellow solid powder

1.2±0.1 g/cm3

475.6±45.0 °C at 760 mmHg

124ºC

241.4±28.7 °C

0.0±1.2 mmHg at 25°C

1.581

2.34

0

5

5

23

473

0

O(C([H])([H])[H])C1C(=C(C([H])=C(C=1[H])/C(/[H])=C(\[H])/C(N1C(C([H])=C([H])C([H])([H])C1([H])[H])=O)=O)OC([H])([H])[H])OC([H])([H])[H]

VABYUUZNAVQNPG-BQYQJAHWSA-N

InChI=1S/C17H19NO5/c1-21-13-10-12(11-14(22-2)17(13)23-3)7-8-16(20)18-9-5-4-6-15(18)19/h4,6-8,10-11H,5,9H2,1-3H3/b8-7+

1-[(E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-2,3-dihydropyridin-6-one

Piperlongumine; 20069-09-4; SGD66V4SVJ; 1-[(E)-3-(3,4,5-trimethoxyphenyl)prop-2-enoyl]-2,3-dihydropyridin-6-one; Piplartine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 63~100 mg/mL (198.5~315.1 mM)
Ethanol: ~63 mg/mL (~198.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (7.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.55 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 4: 2% DMSO+40% PEG 300+2% Tween 80+ddH2O: 5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)