Apoptosis
Periplocin targets the Na/K-ATPase/Src complex receptor. It binds to Na/K-ATPase α1 subunit, which interacts with Src, leading to Src activation and subsequent downstream signaling [1].
Periplocin also targets death receptor 4 (DR4) and Fas-associated death domain (FADD) by inducing their expression, and suppresses inhibitor of apoptosis proteins including cIAP-1, XIAP, and survivin [2].

Treatment of L929 cells with periplocin (5–20 μM) for 48 hours resulted in a 131% increase in proliferation [1]. Periplocin dose-diagnoses and time-promotes fibroblast migration [1]. Periplocin (5 – 20 μM; 30-120 minutes; L929 cells) significantly promotes fibroblast migration. L929 is increased by periplocin (5–20 μM; 48 hours) Periplocin induces PI3K/Akt and Src/ERK activation via Na/KATPase [1].

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Periplocin (5, 10, 20 μM) significantly promoted proliferation of L929 mouse fibroblasts in a concentration-dependent manner after 48 h treatment, as measured by MTT assay (cell viability increased up to 131% at 20 μM, p<0.001) and EdU incorporation assay (percentage of EdU-positive cells increased from 31% to 48% at 20 μM, p<0.001) [1].
Periplocin (5, 10, 20 μM) promoted migration of L929 cells in scratch assay after 48 h, with wound area reduced to 30% at 20 μM compared to control (p<0.001), and in transwell assay after 12 h, the number of migrated cells increased to 156% at 20 μM (p<0.001) [1].
Periplocin (5, 10, 20 μM) increased soluble collagen production in L929 fibroblasts after 48 h, reaching 101 μg/ml at 20 μM compared to 46 μg/ml in untreated group (p<0.001) [1].
Periplocin (5, 10, 20 μM) activated Src/ERK and PI3K/Akt pathways in L929 cells as shown by increased phosphorylation of Src, ERK, PI3K, and Akt in a dose-dependent and time-dependent manner (peak at 60 min). The maximum increases were 315% for p-Src, 302% for p-PI3K, 309% for p-Akt, and 331% for p-ERK at 20 μM (p<0.01) [1].
Knockdown of Na/K-ATPase α1 expression using shRNA lentivirus (mNK1) in L929 cells abolished Periplocin-induced activation of Src/ERK and PI3K/Akt pathways, and also prevented the proliferative and migratory effects of Periplocin [1].
Co-immunoprecipitation and co-immunostaining assays confirmed that Na/K-ATPase α1 subunit interacts with Src in L929 cells [1].
Periplocin (CP-1) inhibited the growth of HA22T/VGH hepatocellular carcinoma cells with an IC50 of 0.027 μM (alone) and showed enhanced cytotoxicity when combined with TRAIL (100 ng/ml) [2].
Periplocin (0.03, 0.1, 0.3, 1, 3, 10 μM) with or without TRAIL (100 ng/ml) for 48 h showed that cotreatment strongly enhanced growth inhibitory activity in HA22T/VGH cells [2].
Periplocin (0.3, 1 μM) combined with TRAIL (100 ng/ml) induced apoptosis in HA22T/VGH cells as shown by Annexin V/PI staining (increased Annexin V and PI positive cells) and sub-G1 population analysis (dose-dependent increase) [2].
Periplocin (1 μM) induced DR4 and FADD expression in HA22T/VGH cells after 8 h treatment, but did not induce DR5 expression [2].
Combination of Periplocin (1 μM) and TRAIL (100 ng/ml) for 24 h increased cleavage of BID, caspase-8, caspase-3, and PARP in HA22T/VGH and Huh-7 cells as shown by western blot [2].
Caspase inhibitors (Z-DEVD-FMK for caspase-3, Z-IETD-FMK for caspase-8, Z-LEHD-FMK for caspase-9, and Z-VAD-FMK for pan-caspase) partially or completely rescued cell viability suppressed by Periplocin and TRAIL treatment in HA22T/VGH cells [2].
Combination of Periplocin (1 μM) and TRAIL (100 ng/ml) for 24 h repressed expression of cIAP-1, XIAP, and survivin in HA22T/VGH cells, but did not affect Bcl-2, Bax, Bad, Mcl-1, or apaf-1 expression [2].
Periplocin dose-dependently repressed cyclin-D1 expression in Huh-7 cells after 24 h treatment as shown by immunofluorescence [2].

Hepatocellular carcinoma (HCC) growth is inhibited in a mouse xenograft tumor model by Periplocin (5–20 mg/kg; intraperitoneal injection; daily; for 14 days; female SCID mice) treatment [2].

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In a rat excisional wound model, Periplocin (5, 10, 20 μg/ml in olive oil, 50 μl once daily for 9 days) significantly accelerated wound closure. On day 6, wound closure was ~72% in the 20 μg/ml group vs. 38% in vehicle control; on day 9, wound area decreased to 7% in the 20 μg/ml group vs. 34% in control (p<0.001). Histological analysis (H&E and Masson's trichrome staining) on day 9 showed increased reepithelization, granulation tissue formation, fibroblast proliferation, and collagen deposition in Periplocin-treated wounds. Periplocin also reduced mononuclear inflammatory infiltrate and lowered IL-1β and TNF-α levels in wound tissue (day 5) [1].
In a xenograft tumor model, SCID mice bearing Huh-7 subcutaneous tumors were treated with Periplocin intraperitoneally (IP) daily for 14 days (5 mg/kg on days 15-29, then 20 mg/kg on days 29-35). Periplocin inhibited tumor growth with tumor growth inhibition (TGI) of 51±11% after 24 days of treatment (p<0.05). Immunohistochemistry showed that Periplocin significantly reduced Ki67-positive cells (57.3±0.67% in vehicle vs. 22.78±10.09% in treated) and cyclin-D1-positive cells (76.87±2.93% in vehicle vs. 58.85±5.05% in treated) in tumor samples [2].

Cell Viability Assay[1]
Cell Types: L929 cells
Tested Concentrations: 5 μM, 10 μM, 20 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Proliferation increased up to 131% at 20 μM.

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Western Blot Analysis[1]
Cell Types: L929 Cell
Tested Concentrations: 5 μM, 10 μM, 20 μM
Incubation Duration: 30 minutes, 60 minutes, 120 minutes
Experimental Results: Src, ERK, PI3K and Akt phosphorylation of the active site were dose-dependent Sex increases in a time-dependent manner.

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For proliferation assay, L929 mouse fibroblasts were seeded into 96-well plates at 5000 cells/well, serum-starved for 24 h, then treated with Periplocin (5, 10, 20 μM) for 48 h. MTT solution (0.5 mg/ml) was added for 3 h, formazan dissolved in DMSO, and absorbance measured at 570 nm. EdU incorporation: cells treated with Periplocin for 24 h, then incubated with 10 μM EdU for 4 h, fixed, permeabilized, reacted with Click-iT mixture, stained with Hoechst 33342, and imaged under fluorescence microscope [1].
For migration assay (scratch), L929 cells grown to 90% confluence in culture plates were serum-starved for 24 h, a linear wound was created with a pipette tip, and cells were treated with Periplocin (5, 10, 20 μM) or Src inhibitor (1 μM). Images were taken at 0 and 48 h, and wound closure area was quantified. Transwell migration: L929 cells (5×10^4 cells/well) in serum-free medium with Periplocin were placed in upper chamber (8.0 μm pore size), lower chamber contained DMEM, after 12 h cells on upper side were removed, invaded cells fixed in cold methanol, stained with crystal violet, and counted [1].
Collagen production assay: L929 cells (1×10^5 cells/well) in 24-well plates were serum-starved for 24 h, then treated with Periplocin (5, 10, 20 μM) for 48 h. Supernatant (100 μl) was mixed with Sircol Dye Reagent for 30 min, centrifuged, pellet re-dissolved in Alkali Reagent, and absorbance measured at 540 nm [1].
Western blot: total protein extracted with RIPA lysis buffer, quantified by BCA, separated by SDS-PAGE, transferred to PVDF membranes, blocked, incubated with primary antibodies against p-Src, p-ERK, p-PI3K, p-Akt, total proteins, and β-actin, then with HRP-conjugated secondary antibody, visualized by ECL [1].
For viability assay (MTT) in HCC cells, HA22T/VGH or Huh-7 cells were seeded at 10^4 cells/well in 96-well plates, treated with Periplocin (0.03-10 μM) with or without TRAIL (100 ng/ml) for 48 h, MTT (5 mg/ml) added to final 0.5 mg/ml for 1-2 h, then 10% SDS added, and absorbance read at 570 nm [2].
Apoptosis quantification by Annexin V/PI: HA22T/VGH cells treated with Periplocin alone or with TRAIL for 24 h, then stained with Annexin V and propidium iodide, analyzed by flow cytometry. Sub-G1 analysis: cells fixed in 70% ethanol, stained with propidium iodide after RNase A treatment, and analyzed by flow cytometry [2].
FACS analysis for surface receptors: cells incubated with dye-labeled monoclonal antibodies against DR4, DR5, DcR1, DcR2 for 30 min on ice, washed, and analyzed by flow cytometry [2].
Immunofluorescence: Huh-7 cells seeded on slides, treated with Periplocin for 24 h, fixed, stained with Hoechst 33258, Ki67, and cyclin-D1 antibodies [2].

Animal/Disease Models: Female SCID (severe combined immunodeficient) mouse (6-8 weeks old) injected with Huh-7 cells [2]
Doses: 5 mg/kg, 20 mg/kg
Route of Administration: intraperitoneal (ip) injection; daily; continued for 14 days
Experimental Results: Inhibition Growth of hepatocellular carcinoma (HCC) in mouse xenograft tumor models.

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For wound healing study: Thirty-five male Sprague-Dawley rats (260-300 g) were anesthetized with chloral hydrate (350 mg/kg), and two full-thickness skin wounds (8 mm diameter) were created on the dorsal area. Rats were randomly divided into five groups and treated with 50 μl of either vehicle (olive oil) or Periplocin in olive oil (5, 10, 20 μg/ml) once daily for 9 days. Recombinant bovine basic fibroblast growth factor (rb-FGF) hydrogel served as positive control. Wound pictures were taken on days 0, 3, 6, 9 and wound area quantified. On day 9, animals were sacrificed, wound tissue collected for histology (H&E and Masson's trichrome staining) [1].
For xenograft tumor model: Female SCID mice (6-8 weeks old) were subcutaneously injected with Huh-7 cells (3×10^6 cells/mouse in equal volumes of PBS and Matrigel) into the right flank. Tumors were allowed to grow to 100-200 mm^3. Periplocin (5-20 mg/kg) or vehicle control (10% NMP, 20% Cremophor EL, 70% saline) was intraperitoneally (IP) injected once daily for 14 days (5 mg/kg on days 15-29, then 20 mg/kg on days 29-35). Tumor volume (V = L×S^2/2) and body weight were measured twice weekly. At study end, mice were sacrificed, tumor samples collected for histology and immunohistochemistry (Ki67 and cyclin-D1 staining) [2].

Periplocin showed low toxicity to normal cells: PBMC cell viability was >80% when treated with 300 μg/ml Periplocin [2].
In the rat wound healing study, no apparent toxicity was reported [1].
In the xenograft study, mice treated with Periplocin at 20 mg/kg IP daily showed a slight decrease in body weight (kept at around 90% of control group), but no further body weight loss was observed, indicating tolerable side effects [2].

[1]. Periplocin promotes wound healing through the activation of Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase. Phytomedicine. 2019 Apr;57:72-83.

[2]. Antitumor Effect of Periplocin in TRAIL-Resistant Human Hepatocellular Carcinoma Cells through Downregulation of IAPs. Evid Based Complement Alternat Med. 2013;2013:958025.

Periplocin has been reported to be found in Periploca sepium, Periploca forrestii, and other organisms with available data.

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Periplocin is a cardiotonic steroid and the most abundant compound in Periploca forrestii. It promotes wound healing by activating Src/ERK and PI3K/Akt pathways mediated by Na/K-ATPase/Src complex. The interaction between Na/K-ATPase α1 subunit and Src was confirmed by co-immunoprecipitation and co-immunostaining in L929 cells. Silencing Na/K-ATPase α1 abolished the wound healing effects of Periplocin [1].
Periplocin resensitizes TRAIL-resistant hepatocellular carcinoma cells to TRAIL-induced apoptosis through two mechanisms: (1) inducing DR4 and FADD expression, and (2) suppressing IAPs (cIAP-1, XIAP, survivin), leading to activation of caspase-8, -9, -3 and PARP cleavage. The combination of Periplocin and TRAIL synergistically induces apoptosis in TRAIL-resistant HCC cells [2].
Structurally, Periplocin (CP-1) differs from periplogenin (CP-5) by one disaccharide residue, and the saccharide residue may affect protein interactions [2].

C36H56O13

696.82

682.356

13137-64-9

14463159

White to off-white solid

1.4±0.1 g/cm3

877.4±65.0 °C at 760 mmHg

205°C

272.8±27.8 °C

0.0±0.6 mmHg at 25°C

1.624

-1.36

6

13

7

49

1280

17

O[C@]12CC[C@H](C3=CC(=O)OC3)[C@@]1(C)CC[C@]1([H])[C@@]3(C)CC[C@@H](C[C@]3(CC[C@@]21[H])O)O[C@H]1C[C@@H]([C@@H]([C@@H](C)O1)O[C@H]1[C@@H]([C@H]([C@@H]([C@@H](CO)O1)O)O)O)OC

KWBPKUMWVXUSCA-AXQDKOMKSA-N

InChI=1S/C36H56O13/c1-18-31(49-32-30(41)29(40)28(39)25(16-37)48-32)24(44-4)14-27(46-18)47-20-5-9-33(2)22-6-10-34(3)21(19-13-26(38)45-17-19)8-12-36(34,43)23(22)7-11-35(33,42)15-20/h13,18,20-25,27-32,37,39-43H,5-12,14-17H2,1-4H3/t18-,20+,21-,22+,23-,24+,25-,27+,28-,29+,30-,31-,32+,33-,34-,35+,36+/m1/s1

3-[(3S,5S,8R,9S,10R,13R,14S,17R)-5,14-dihydroxy-3-[(2R,4S,5R,6R)-4-methoxy-6-methyl-5-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-10,13-dimethyl-2,3,4,6,7,8,9,11,12,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-17-yl]-2H-furan-5-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~100 mg/mL (~143.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.59 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 (3.59 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 25.0 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 3: ≥ 2.5 mg/mL (3.59 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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 (Please use freshly prepared in vivo formulations for optimal results.)