The data in this article are related to the research article entitled “Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations” Botezatu et al. [1]. Somatic mutations in the PIK3CA gene (“hot spots” in exons 9 and 20) are found in many human cancers, and their presence can determine prognosis and a treatment strategy. An effective method of mutation scanning PIK3CA in clinical laboratories is DNA Melting Analysis (DMA) (Vorkas et al., 2010; Simi et al., 2008) [2], [3]. It was demonstrated recently that the TaqMan probes which have been long used in Real Time PCR may also be utilized in DMA (Huang et al., 2011) [4]. After optimization of this method Botezatu et al. [1], it was used for multiplex scanning PIK3CA hotspot mutations in formalin-fixed paraffin-embedded (FFPE) samples from patients with colorectal and lung cancer.
Asymmetric PCR and DNA melting analysis with TaqMan probes applied for mutation detection is effectively used in clinical diagnostics. The method is simple, cost-effective, and carried out in a closed-tube format, minimizing time, labor, and risk of sample cross-contamination. Although DNA melting analysis is more sensitive than Sanger sequencing (mutation detection thresholds are ~5% and 15%-20%, respectively), it is less sensitive than more labor-intensive and expensive techniques such as pyrosequencing and droplet digital PCR. Here, we demonstrate that, under specially selected conditions of asymmetric PCR, TaqMan probes can play the role of blocking agents. Preferential blocking of the wild-type allele brings about enriched amplification of mutant alleles. As a result, an ~10-fold increase in the detection sensitivity for mutant and genes was achieved.
The hormonal compound with the highest cytostatic activity against MCF-7 tumor cells (human breast cancer, BC) and the lowest activity against normal cells (rat skin fibroblasts) was sought among gestagens, androstenes, and antiestrogencytostatics. It was found that antiestrogencytostatics and androstenes had the highest cytostatic activity against tumor cells whereas gestagens and antiestrogencytostatics were least active against fibroblasts. Studies of the activity of the hormonal compounds in combination with doxorubicin on the viability of MCF-7 and rat skin fibroblasts found that all investigated compounds with the exception of dehydroepiandrosterone (DHEA) intensify the cytostatic activity of doxorubicin against tumor cells, the greatest effect seen for antiestrogencytostatics. A chemoprotective effect of androstenes on normal cells was noted.Treatment regimes for cancerous diseases have not yet been standarized despite the efforts of researchers and clinicians. This is apparently due to the fact that each tumor has its own receptor array and individual genetic profile and responds differently to one or another type of therapy. Agonists or antagonists of various steroidal receptors have turned out in several instances to be highly effective in treating hormone -dependent neoplasms. Depending on the age of the patient (reproductive or menopausal), various combinations of antiestrogens, progestins, and even androgens may be used in hormone therapy of human breast cancer (BC) [1]. Herein we report the screening of new synthetic gestagens, antiestrogencytostatics, and androstenes and the comparison of them with existing clinical drugs for tumor model and normal cells. The existing clinical drugs have several side effects and drawbacks (multidrug resistance and glucocorticoid, mineralocorticoid, and androgen side effects of hormone -therapy). Therefore, there is a continuous search for more effective and safer new compounds. Several new steroidal compounds have been synthesized. We studied the following of these: 1) 17a-Acetoxy-3b-butyloxy-6-methylpregna-4,6-dien-20-one (ABMP), a gestagen that does not have a D 4 -3-ketone and has a structure similar to progesterone but, in contrast with it, can be readily absorbed orally [2]. The reference drugs were progesterone (P) and medroxyprogesterone acetate (MPA).2) Po715 and Po716, antiestrogencytostatics that are estrogens having transformed C rings with substituents containing a bis-b-chloroethylamine moiety (Fig. 1) [3,4].We used 11a-derivatives of ethynylestradiol with the bis-b-chloroethylamine moiety bonded to the 3 -position.3) Dehydroepiandrosterone nitrate (DHEAN), androstendiol nitrate (AEDN), and androstendiol dinitrate (AEDDN), which are nitro derivatives of the endogenous androstenes dehydroepiandrosterone and androstendiol, respectively. The reference drugs were androstendiol (AED) and DHEA [5].
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