Saponins From Paris forrestii (Takht.) H. Li Display Potent Activity Against Acute Myeloid Leukemia by Suppressing the RNF6/AKT/mTOR Signaling Pathway

Saponins are amphipathic glycosides found in traditional Chinese medicines. In the present study, we isolated a panel of saponins from Paris forrestii (Takht.) H. Li, a unique plant found in Tibet and Yunnan provinces, China. By examining their activities in suppressing acute myeloid leukemia (AML) cell proliferation, total saponins from Paris forrestii (TSPf) displayed more potent activity than individual ones. TSPf induced more than 40% AML cell apoptosis and decreased the viability of all leukemia cell lines. TSPf-induced apoptosis was confirmed by both Annexin V staining and caspase-3 activation. In line with these findings, TSPf downregulated pro-survival proteins Mcl-1, Bcl-xL, and Bcl-2 but upregulated the expression of tumor suppressor proteins p53, p27, Bax, and Beclin 1. The AKT/mTOR signaling pathway is frequently overactivated in various AML cells, and TSPf was found to suppress the activation of both AKT and mTOR, but had no effects on their total protein expression. This was further confirmed by the inactivation of 4EBP-1 and p70S6K, two typical downstream signal molecules in the AKT/mTOR pathway. Moreover, TSPf-inactivated AKT/mTOR signaling was found to be associated with downregulated RNF6, a recently identified oncogene in AML. RNF6 activated AKT/mTOR, and consistently, knockdown of RNF6 led to inactivation of the AKT/mTOR pathway. Furthermore, TSPf suppressed the growth of AML xenografts in nude mice models. Oral administration of TSPf almost fully suppressed tumor growth without gross toxicity. Consistent with the findings in cultured cell lines, TSPf also downregulated RNF6 expression along with inactivated AKT/mTOR signaling in tumor tissues. This study thus demonstrated that TSPf displays potent anti-AML activity by suppressing the RNF6/AKT/mTOR pathway. Given its low toxicity, TSPf could be developed for the treatment of AML

The association between exposure to PM2.5 components from coal combustion and mortality in female breast cancer patients

PM _2.5 components may promote the development of breast cancer and increase the risk of mortality. This study aims to investigate the associations between long-term exposure to PM _2.5 components and multiple causes of mortality among women with breast cancer living in Inner Mongolia, China. We constructed an Inner Mongolia cohort of 33 952 breast cancer patients from 2012 to 2021 using data from the Inner Mongolia Regional Health Information Platform. We assessed each patient’s exposure to PM _2.5 components using the Tracking Air Pollution in China database. Cox regression models were used to estimate adjusted hazard ratios and 95% confidence intervals (95% CIs). A total of 3295 deaths were identified. For each interquartile increase in concentration in the 5 years before diagnosis, the all-cause mortality increased significantly by 5% (HR: 1.05, 95%CI: 1.00–1.10) for black carbon and by 4% (HR: 1.04, 95%CI: 1.00–1.09) for sulfate (SO _4 ^2− ), and decreased by 7% (HR: 0.93, 95%CI: 0.88–0.98) for nitrate (NO _3 ^− ). An association between organic matter and an increased all-cause mortality was also observed. Similar results were found for associations with risk of death from breast cancer-specific causes, cardio-cerebrovascular disease (CCVD) causes, and respiratory causes. Stronger associations were observed in older age groups and in Han Chinese patients. Our results showed that long-term exposure to black carbon, organic matter, and SO _4 ^2− were more responsible for the increased risk of death from all causes, breast cancer-specific causes, CCVD causes, and respiratory causes. This suggests that more effective measures to control coal combustion emissions in Inner Mongolia are urgently needed. The elderly and Han Chinese populations may be at high risk

CLCuMuB βC1 Subverts Ubiquitination by Interacting with NbSKP1s to Enhance Geminivirus Infection in <i>Nicotiana benthamiana</i>

<div><p>Viruses interfere with and usurp host machinery and circumvent defense responses to create a suitable cellular environment for successful infection. This is usually achieved through interactions between viral proteins and host factors. Geminiviruses are a group of plant-infecting DNA viruses, of which some contain a betasatellite, known as DNAβ. Here, we report that <i>Cotton leaf curl Multan virus</i> (CLCuMuV) uses its sole satellite-encoded protein βC1 to regulate the plant ubiquitination pathway for effective infection. We found that <i>CLCuMu betasatellite</i> (CLCuMuB) βC1 interacts with NbSKP1, and interrupts the interaction of NbSKP1s with NbCUL1. Silencing of either <i>NbSKP1s</i> or <i>NbCUL1</i> enhances the accumulation of CLCuMuV genomic DNA and results in severe disease symptoms in plants. βC1 impairs the integrity of SCF^COI1 and the stabilization of GAI, a substrate of the SCF^SYL1 to hinder responses to jasmonates (JA) and gibberellins (GA). Moreover, JA treatment reduces viral accumulation and symptoms. These results suggest that CLCuMuB βC1 inhibits the ubiquitination function of SCF E3 ligases through interacting with NbSKP1s to enhance CLCuMuV infection and symptom induction in plants.</p></div

The N-terminal domain of NbSKP1.1 interacts with CLCuMuB βC1 in yeast.

<p>Growth of SKY48 yeast strains containing NLS-LexA BD-CLCuMuB βC1 (BD-βC1) transformed with AD fused full length, N-terminal fragment (N98aa), C-terminal fragment (C57aa) of NbSKP1.1 or AD (control) on Leu-containing (Leu⁺) and Leu-deficient (Leu⁻) medium with galactose (Gal) and raffinose (Raf) at 28°C for 6 d. Yeast cells were plated at OD₆₀₀ = 1, 0.1, 0.01.</p

CLCuMuB βC1 hinders the degradation of YFP-GAI <i>in vivo</i>.

<p>(A) CLCuMuB βC1 attenued degradation of YFP-GAI <i>in vivo</i>. YFP-GAI expression construct was coinfiltrated with constructs expressing HA-nLUC or HA-βC1 into seven to eight-week-old <i>N</i>. <i>benthamiana</i> plant leaves. Around 48 hpi, agroinfiltrated leaves were sprayed with 100 μM GA₃ or mock solution (ethonal) and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scales represents 200 μm. DMSO and MG132 (50 μM) were applied into plant leaves 12 h before observation. Protein level was analyzed via SDS-PAGE and western blot analysis with the anti-GFP antibody, which also recognizes YFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control. (B) Real-time RT-PCR detected the mRNA level of YFP-GAI. Total RNA was extracted from each <i>N</i>. <i>benthamiana</i> leaves and then subjected to quantitative RT-PCR (means±SEM, n = 3) to quantify YFP-GAI mRNA level. <i>eIF4a</i> was used as the internal reference. (C) CLCuMuB βC1 didn’t affect stability of GFP <i>in vivo</i>. Detection of GFP (as an internal control) in <i>N</i>. <i>benthamiana</i> leaves coinfiltrated with the construct expressing GFP together with constructs expressing HA-nLUC or HA-βC1 and treated with 100 μM GA₃ or mock (ethanol) solution and visualized via a Zeiss LSM 710 laser scanning microscope. Bar scale represents 200 μm. Protein level was analyzed via SDS-PAGE and immunoblot analysis with anti-GFP. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as a loading control.</p

Silencing of <i>NbSKP1s</i> enhances CLCuMuV DNA accumulation and results in typical disease symptoms.

<p>(A1, A2 and A3) Six- to seven-week-old <i>N</i>. <i>benthamiana</i> plants were agroinoculated with CLCuMuV (CA) and βM2-<i>SKP1</i>F1 (A1), βM2-<i>SKP1</i>F2 (A2), βM2-<i>SKP1</i>F3 (A3) or βM2-<i>βC1</i>F (as the control). (B1, B2 and B3) Silencing of <i>NbSKP1s</i> enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1, C2 and C3) Real-time RT-PCR confirmed silencing of <i>NbSKP1s</i>. Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify <i>NbSKP1s</i> mRNA level. <i>Actin</i> was used as the internal reference. The raw data of (B1–B3) and (C1–C3) were analysed by two-sample <i>t</i>-test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1, D2 and D3) 50% plants infected with CA+βM2-<i>SKP1</i>F1 (D1), 50% plants infected with CA+βM2-<i>SKP1</i>F2 (D2) and 100% plants infected with CA+βM2-<i>SKP1</i>F3 (D3) show severe symptoms at 21 dpi. (E1, E2, E3 and E4) Apical leaves of plants infected with CA+βM2-<i>βC1</i>F (E1), CA+βM2-<i>SKP1</i>F1(E2), CA+βM2-<i>SKP1</i>F2 (E3) and CA+βM2-<i>SKP1</i>F3 (E4) at 21 dpi.</p

CLCuMuB βC1 interferes with the interaction between NbCUL1 and NbSKP1.1 <i>in vitro</i> and <i>in vivo</i>.

<p>(A) GFP competitive pull-down assay <i>in vitro</i>. His-βC1 was expressed in <i>E</i>. <i>coli</i> as inclusion body and refolded through urea-arginine dialysis. BSA (NEB, USA) was used as a control. GFP-NbCUL1 or GFP was expressed in <i>N</i>. <i>benthamiana</i> leaves and trapped through GFP-Trap agarose. After the supernatant was discarded, GFP-Trap agarose was incubated with <i>E</i>. <i>coli</i>-expressed His-HA-NbSKP1.1, then the supernatant was discarded. GFP-Trap agarose was incubated with gradient dilutions (1, 1/2, 1/4) of His-βC1. Finally, agarose was washed and proteins were analyzed via SDS-PAGE and western blot assays using anti-GFP and anti-HA antibodies. Input was analyzed by the anti-His antibody (EASYBIO, China) and supernatant was analyzed by the anti-HA antibody. Intensity was detected through Total Lab TL120. (B) A confocal image of BiFC assays show that CLCuMuB βC1 interfered with the interaction between NbCUL1 and NbSKP1.1 <i>in vivo</i>. Photos were taken at 48 hpi. Bar scale represents 200 μm. (C) BiFC intensity (means±SEM, n = 4) was quantified by YFP fluorescence. Relative BiFC intensity was normalized to the control. The raw data were analyzed by two-sample <i>t</i>-test to show the significance level at 0.01 (**). (D) The protein level of cYFP-NbCUL1 and nYFP-NbSKP1.1 were checked with the polyclonal GFP antibody (Huaxin Bochuang, China). The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as the loading control.</p

Silencing of <i>NbCUL1</i> enhances CLCuMuV DNA accumulation and results in typical disease symptoms.

<p>(A1 and A2) Six- to seven-week-old <i>N</i>. <i>benthamiana</i> plants were agroinoculated with CLCuMuV (CA) and βM2-<i>CUL1</i>F1 (A1), βM2-<i>CUL1</i>F2 (A2) or βM2-<i>βC1</i>F (as the control). (B1 and B2) Silencing of <i>NbCUL1</i> enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1 and C2) Real-time RT-PCR confirmed silencing of <i>NbCUL1</i>. Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify <i>NbCUL1</i> mRNA level. <i>Actin</i> was used as the internal reference. The raw data of (B1 and B2) and (C1 and C2) were analysed by two-sample <i>t</i>-test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1 and D2) 100% plants infected with CA+βM2-<i>CUL1</i>F1 (D1) or CA+βM2-<i>CUL1</i>F2 (D2) show severe symptoms at 21 dpi.</p

CLCuMuB βC1 represses JA responses though interfering with the integrity of SCF^COI1.

<p>(A) Total root length of HA-βC1 transgenic (#2 HA-βC1 and #3 HA-βC1) and wild-type (#2 WT and #3 WT) <i>N</i>. <i>benthamiana</i> seedlings was measured every 24 h beginning at the 6th day after sowing (n ≥11). Bars represent SEM. #2 HA-βC1 and #2 WT were presented on same plates, while #3 HA-βC1 and #3 WT were presented on same plates. These experiments were repeated 3 times. (B) Jasmonate sensitivity was measured as root growth inhibition. Six-day-old seedlings (n ≥10) were grown on MS contained with 50 μM MeJA for additional 4 days. Bars represent SEM. The raw data were analysed by Mann-Whitney rank sum test to show the significance level at 0.05 (*). (C) Relative expression level of marker genes of jasmonate responses in mock- or MeJA-treated HA-βC1 transgenic and wild-type <i>N</i>. <i>benthamiana</i> seedlings determined by quantitative real-time PCR. #2 HA-βC1 and #2 WT were presented on same plates, while #3 HA-βC1 and #3 WT were presented on same plates. HA-βC1-expressing lines are compared with their corresponding control in each condition. <i>Actin</i> was used as the internal control. Bars represent SEM. The raw data were analysed by two-sample <i>t</i>-test to show the significance level at 0.05 (*), 0.01 (**) and 0.001 (***). These experiments were repeated at least twice. (D) CLCuMB βC1 enhanced degradation of COI1 <i>in vitro</i>. The purified Myc-COI1 protein was added to total protein extracts from <i>N</i>.<i>benthamiana</i> which expressed HA-nLUC or HA-βC1, incubated at 25°C for the indicated time periods, and subjected to immunoblot analysis with the anti-Myc antibody. Intensity was detected through Total Lab TL120. The PVDF membrane was stained with Ponceaux to visualize the large subunit of ribulose-1,5-bisphosphate as the loading control. (E) Quantitative analysis of the relative abundance of COI1 in the presence of HA-nLUC or HA-βC1 for the time periods indicated. The abundance of COI1 at the start point (0-h) was set to 100% as a reference for calculating its relative abundance after different incubation periods. Error bars represent SD. The experiment was repeated three times.</p